| Background and ObjectiveCervical Cancer(CC)is currently the fourth most common malignant disease of women worldwide.Although the incidence and mortality of cervical cancer decreased with screening of precancerous and the development of new treatment strategies,a significant number of metastatic or recurrent disease is still diagnosed.For those people not suitable for curative treatments,such as surgery and radiation,palliative chemotherapy remains the main treatment.Because of the limited therapeutic effect of chemotherapeutic drugs,the development of new biomolecule targeted therapy is one of the hotspots in recent years.Targeted anti-tumor therapy for ?1 receptor has attracted the attention of researchers.?1 receptor(?1R)is a class of non-opioid transmembrane protein,distributed in a variety of organ tissues in mammals include brain,fundus,cornea,liver,vascular cells and various tumors.The ?1R protein is mainly located in the mitochondria-associated endoplasmic reticulum(MAM),which can be translocated to the cell membrane and nuclear membrane under stimulation of the agonist or stress.?1R has been demonstrated to be a ligand-regulated chaperone protein,involved in physiology and pathology of various human diseases,such as neurodegenerative diseases(such as Parkinson's disease),mental disorders(such as depression)and central and peripheral tumors.Highly selective ligand drugs for ?1R have potential for the diagnosis and treatment of the receptor-related diseases.The present study examined the expression of ?1R in cervical cancer cell lines and analyzed the relationship between ?1R mRNA level and the clinicopathological features and prognosis of cervical cancer patients in TCGA(The Cancer Genome Atlas),and detected the changes of ?1R protein in plasma of pre-and postoperative cervical cancer in order to explore the value of ?1R in the clinical diagnosis,treatment and prognosis of cervical cancer.On the other hand,specific small molecule ligand compounds for ?1R were synthesized and used to detect its effect on biology of cervical cancer cell line,providing evidences for new molecular targeted diagnosis and therapy for cervical cancer.Materials and MethodsImmunocytochemical staining was used to detecte the localization and expression of ?1R protein in cervical cancer cells.The ?1R RNA-seqV2 data of the pretreated cervical cancer data set was downloaded from the TCGA database(https://genome-cancer.cs.ucsc.edu/),leaving only cases which contained clinical parameters and survival data.The expression of ?1R in different clinicopathological parameters was compared by Student's t test.Survival analysis of cervical cancer was carried out by Kaplan-Meier.The changes of ?1R protein in plasma of pre-and postoperative cervical cancer were detected by Western blotting.The effects of four novel ?1R-specific ligand compounds on the biology of HeLa and Siha in cervical cancer were examined: MTT assay was used to detect the inhibitory effect of ligand compounds on the growth and the sensitivity of nedaplatin of HeLa and Siha cells.Cell scratches were used to observe the effect of ligand compounds on cell migration ability.Flow cytometry was used to examine the effect on the cell cycle and apoptosis rate.Results1.Immunocytochemical staining showed that ?1R was expressed in cervical cancer HeLa and Siha cells,and located in the cytoplasm and nucleus.2.The expression of ?1R in squamous cell carcinoma was correlated with age(P=0.005).The expression of ?1R in adenocarcinoma was significantly correlated with clinical stage(P=0.020),lymph node metastasis(P=0.004),vascular invasion(P=0.018).High expression of ?1R had lower median survival [(35.5±10.5)months)] than that of low expression [(95.4±15.0)months](P<0.05)in adenocarcinoma.3.Western blot analysis showed that the level of ?1R in plasma of patients with 10 days after operation was significantly lower than that before operation(P<0.001).4.MTT showed that novel four compounds inhibited cell proliferation in time and concentration-dependent manner(all P<0.001).IC50 values of compounds 14 a,14e,15 c and 15 f for HeLa and Siha cells were respectively 37.2,41.6,133.7 and 28.0?mol / L and 26.8,28.4,112.9,and 13.8?mol / L.5.Effects on sensitivity to nedaplatin: 6?mol/L 14 a combined with different concentrations of NDP on cells for 48 h,for HeLa cell,the inhibitory rate of the combination of 0.1~10?g/ml NDP were significantly higher than that of the NDP alone group(P<0.01);for Siha cell: the inhibitory rate of the combination of 0.1~30?g/ml NDP were significantly higher than that of the NDP alone group(P<0.01).6.Effects on cell migration: 30?mol/L of 14 a on HeLa and Siha cells for 24 h,the mobility of the experimental group were(19.6 ± 0.6)%,(15.8 ± 0.8)%,which were lower than those in the control group(43.0 ± 1.2)%,(29.3 ± 1.0)%(all P <0.01)7.Effects on cell cycle and apoptosis: Flow cytometry showed that after HeLa and Siha cell treated with compound 14a(30?mol/L)for 48 hours,the total apoptotic rates of HeLa and Siha cells were(16.30±1.13)% and(21.27±1.68)% respectively,which were higher than those of the untreated group(1.80±0.27)%,(2.75±0.21)%(PHeLa=0.032,PSiha=0.042).The percentage of G0/G1 phase cells in compound group was significantly increased(PHeLa=0.032,PSiha=0.042).Conclusions1.?1R is expressed in the cytoplasm and nucleus of cervical cancer HeLa and Siha cell lines.2.The high expression of ?1R in cervical adenocarcinoma is closely related to tumor invasion,progression,and poor prognostic factor.?1R level in plasma might be a biomarker for monitoring the therapeutic effect of cervical cancer.3.The novel synthesized ligand compounds for ?1R significantly inhibit the proliferation of cervical cancer HeLa and Siha cells in a concentration and time-dependent manner.4.?1R ligand compound 14 a inhibits the migration of HeLa and Siha cells.5.?1R ligand compound 14 a significantly increases the sensitivity of cervical cancer cells to nedaplatin at low concentrations.6.?1R ligand compound 14 a may play anti-tumor effect through the apoptosis-inducing pathway and the cell cycle G1 phase arrest. |
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