The Effect Of ?1 Receptor Intervention On Sensitization Of Cervical Cancer Cell Line To TRAIL Inducer

Background and ObjectiveCervical cancer is the one of the most common gynecological malignant tumors,which seriously threatens women's health.The incidence of cervical cancer is increasing by years,which is a serious threat to the physical and mental health of patients.The main cause of cervical cancer is the persistent infection of high-risk human papillomavirus(HPV),HPV can affect cell cycle progression,apoptosis and differentiation.It inactivates or degrades tumor suppressor genes and pro-apoptotic proteins,lead to cell carcinogenesis.Traditional surgery radiotherapy,chemotherapy in locally advanced cervical cancer and recurrent cervical cancer patients have not achieved satisfactory results,the latest targeted therapy and immunotherapy have made relatively satisfactory progress.Bring new choices and hope to patients.At the level of cell and molecule,targeted therapy designs corresponding drugs to the known carcinogenic sites.The drug specifically binds to the carcinogenic sites in vivo and causes specific death of tumor cells,but has no killing effect on normal tissue cells.Therefore,the development of more effective targeting drugs are bound to provide more benefits for the treatment of cervical cancer patients.The tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is a kind of factor that can specifically recognize tumor cell death receptor.It can initiate downstream apoptosis process by binding to death receptor domain,and it is highly efficient.Highly selective induction of multiple tumor cell apoptosis has no obvious toxic side effects on normal tissues and cells.TRAIL has a wide range of application prospects due to its specific killing effect on tumor cells.Many clinical studies have shown that cancer cells develop TRAIL resistance and ultimately avoid TRAIL-induced apoptosis.To clarify the mechanism of TRAIL activation and apoptosis is essential for developing strategies to maximize the potential efficacy of trail in clinical applications.?receptor(?R)is a unique non-opioid receptor protein that can be divided into two subtypes?1R and?2R have different tissue distribution,cell function and pharmacological characteristics.?1R is a special transmembrane protein mainly located in mitochondrial endoplasmic reticulum.It can interact with other proteins,such as Nmethyl-D-aspartic acid and opioid receptor,and regulate the synthesis and activity of intracellular proteins.Its ligand antagonists can be used to treat mental illness,pain and induce apoptosis of various tumor cells.?1R expression could significantly decrease the apoptosis of prostate cells induced by TRAIL.High level of?1R makes prostate cancer cells more tolerant to TRAIL therapy,and?1R plays a key role in the activation of caspase induced by TRAIL.?1R can be used as a promising molecule to make human breast cancer sensitive to trail and be a key mediator for TRAIL-induced tumor specific killing.Nowadays some studies show that there is a high expression of?1R in cervical cancer tissues,especially in cervical adenocarcinoma and cervical cancer Siha and Hela cells.?1R ligand compounds could significantly inhibit the growth of cervical cancer cells.To investigate the effects of?1R ligand compound and TRAIL and?1R plasmid shRNA and upregulated plasmid transfection on the proliferation,migration and cloning of cervical cancer Siha and Hela cells.Materials and Methods?1R antagonist BD1063,?1R agonist Avex-73,TRAIL inducer TIC10,MTT colorimetry was used to detect the effect of compounds on cell proliferation,cell scratch test was used to detect the effects of compounds on cell migration,and plate cloning assay was used to detect the effects of compounds on cell clone formation ability.Flow Cytometry is used to detect the effect of compounds on apoptosis.,western blot technique was used to detect the expression of?1R protein and apoptosis protein.The expression of?1R protein and apoptosis protein in Siha and Hela cells were observed after construction of?1R vector plasmid shRNA and upregulated plasmid transfection.Statistical analysisAll data were analyzed by SPSS 22.0 software.For the measured data,the normal test was first carried out.The t test was used for the comparison between the two groups,and the t test for the minimum significant difference(LSD-t test)was used for the comparison between the two groups.One-way ANOVA was used for multi-group comparison.The counting data were expressed by rate and?~2 test was used.There was a significant difference in P<0.05.Results1.MTT showed that BD1063 and TIC10 inhibited the proliferation of Siha and Hela cells in a concentration-and time-dependent manner(P<0.01),and the IC50values of BD1063 were 47.45?mol/L and 38.37?mol/L when Siha and Hela cells were incubated for 48 h,respectively.The IC50 values of TIC10 were 30.86?mol/L and 31.32?mol/L,respectively.2.The effect of 6?mol/L BD1063 combined with different concentrations of TIC10,on cell inhibition was significantly higher than that of TIC10 alone(P<0.01).The sensitizing effect to TRAIL was significantly higher than that of TIC10 alone(P<0.01).3.The effect of compounds on cell migration:after 24 h of treatment with 48?mol/L BD1063 and 40?mol/L TIC10,the migration rate of Siha and Hela cells was significantly lower than that of the control group(P<0.01);4.The effect of 48?mol/L BD1063 and 40?mol/L TIC10 for 2 weeks on clone forming ability was significantly lower than that of the control group(P<0.01),and that of the control group was significantly lower than that of the control group(P<0.01).5.The effect of compound on apoptosis:after 48h of treatment with 48?mol/L BD1063 and 40?mol/L TIC10,the apoptosis rate was higher than that of control group(P<0.01),6.Western blot showed that after BD1063 combined with TIC10,the expression level of?1R decreased and the expression of cleaved caspase-3,cleaved caspase-8protein increased significantly(P<0.05).7.After transfection with shRNA plasmid,the proliferation of Siha and Hela cells was significantly inhibited(P<0.01),and the inhibition rate of Siha and Hela cells was significantly higher than that of TIC10 after 48h treatment with TIC10(P<0.01),932plasmid had the best inhibitory effect(P<0.01).8.BD1063 had no obvious inhibitory effect on the proliferation of HaCat cells,while Avex-73 and?1R up-regulated plasmid had no obvious inhibitory effect on the proliferation of Siha and Hela and HaCat cells(P>0.05).Conclusions1.?1R antagonist BD1063 and TRAIL inducer TIC10 inhibited the proliferation of Siha and Hela cells in a concentration gradient and time dependent manner.2.BD1063 and TIC10 inhibited the migration of Siha and Hela cells;decrease the clone formation rate of Siha and Hela cells;increase the apoptosis of Siha and Hela cells.3.Low concentration of BD1063 increased the sensitivity of TIC10 to Siha and Hela cells.4.The transfection of shRNA plasmid could decrease?1R expression and increase the sensitivity of TIC10 to Siha and Hela cells.