Sufentanil Can Effect The Growth Of Human Lung Adenocarcinoma Cells By Upregulating Opioid Growth Factor Receptor Expression

Objective:At present,the morbidity of lung cancer ranks the second and the mortality ranks the first in the world.In non-small cell lung cancer(NSCLC),which accounts for about85% of total lung cancers,the morbidity of adenocarcinoma(ADC)is higher than that of squamous cell carcinoma(SCC).Patients suffered lung cancer often experience moderate to severe pain during surgery and palliative care.Sufentanil(SUF)is a strong opioid analgesic that can selectively act on the μ opioid receptor(MOR)to play an analgesic effect,and is widely used in perioperative analgesia and cancer pain treatment.A large number of studies have found that opioids and opioid receptors can affect the occurrence and development of tumors.Clinical retrospective studies have found that increasing dosage of opioids in perioperative analgesia and cancer pain treatment can reduce the survival and prognosis of patients.While cell experiments suggested that various concentrations of SUF played different effects on the growth of different tumor cells,and the underlying mechanisms still needed to be further explored.Opioid growth factor receptor(OGFR)is an integral membrane protein associated with the nucleus,which contains a nuclear localization signal,so it can be transported to the nucleus to regulate DNA synthesis,so as to regulate the growth of cells and tissues.Studies have found that morphine can combine with OGFR to inhibit the growth of lung cancer cells,and can be competitively replaced by its specific ligand opioid growth factor.The roles of OGFR can also be blocked by opioid antagonists.There is no research on whether SUF interacts with OGFR to regulate the growth of tumor cells.Therefore,in this study we analyzed the influence of SUF on the malignant biological behaviors of ADC cell lines and the effect of OGFR expression on this regulatory behavior,so as to explore the possible mechanism of SUF affecting tumor cell growth and provide the experimental evidence for the selection of analgesic for lung cancer patients.Methods:1.In this study,three ADC tumor cell lines H1975,H23 and H358 were selected,and the expressions of OGFR and MOR proteins in cells were detected by Western Blot(WB)experiment.The cell lines with higher expression of OGFR and lower expression of MOR were screened.The CCK-8 cell proliferation assay was used to detect the effects of different concentration gradients(0,1.25,2.5,5,10,20,40 μM)of SUF on the proliferation ability of ADC cell lines,and the IC50 concentration of SUF was calculated and the optimal inhibition time was determined.2.EdU method and plate colony formation assay were used to detect the effects of IC50 concentration of SUF on the proliferation and clone ability of ADC cell lines.The effect of SUF on cell apoptosis was observed by FACS method.The expression of apoptotic proteins were detected by WB experiment.Cell scratch assay and transwell invasion assay were used to detect the effect of SUF on the migration and invasion ability of ADC cell lines.The effect of SUF on OGFR expression in ADC cells was detected by WB experiment and immunofluorescence assay.3.shRNA transfection technology was used to silence OGFR expression,and the transfection efficiency was verified by immunofluorescence and WB experiments.ADC cells were divided into 3 groups,blank plasmid group(sh NC),SUF-treated blank plasmid group(sh NC+SUF)and SUF-treated shOGFR plasmid group(shOGFR+SUF).The cloning ability of cells was detected by plate colony formation assay.The apoptosis of cells was detected by FACS method,and the expression of OGFR and apoptotic protein was detected by WB experiment.Results:1.H1975 and H23 cell lines showed higher expression of OGFR(p<0.01;p<0.05)and lower expression of MOR(p<0.05),and H358 cell line showed lower expression of OGFR(p<0.01;p<0.05)and higher expression of MOR(p<0.05).H1975 and H23 cell lines were selected for subsequent experiments.The results of CCK-8 method showed that after treatment of SUF for 48 hours,it effectively inhibited the proliferation of H1975 and H23 cells.The measured IC50 value of SUF was 32.1μM for H1975 cells and 23.6 μM for H23 cells.2.H1975 and H23 cells were treated with IC50 concentration of SUF for 48 hours.EdU experiments showed that SUF could significantly reduce the percentage of EdUpositive cells in H1975 and H23 cells.The results of cloning experiments showed that SUF significantly inhibited the size and number of clonal clusters of H1975 and H23 cells,and the colony formation rates were 42.00%±1.16%(p<0.0001)and31.00%±2.89%(p<0.01)respectively,which was significantly lower than that in the control group.Flow cytometry analysis found that SUF could significantly increase the apoptosis rates of H1975 and H23 cells,which were 40.90%±1.15%(p<0.0001)and36.74%±2.09%(p<0.001)respectively,compared with the control group(6.05%±0.40%).The results of WB experiment showed that SUF could up-regulate the expression of Bax(p<0.001;p<0.0001)and cleaved caspaspe-3(p<0.01;p<0.0001),and down-regulate the expression of Bcl-2 protein(p<0.001;p<0.0001)in H1975 and H23 cells.The results of the cell scratch test showed that SUF treatment for 24 hours could significantly reduce the scratch healing rate of H1975 and H23 cells(p<0.01),and reduce more in the H23 cell line 48 hours after SUF treatment(p<0.001).The results of transwell experiments showed that SUF significantly reduced the number of invasive cells in H1975 cells(p<0.0001)and H23 cells(p<0.01).The results of WB and immunofluorescence assays showed that the expression of OGFR was increased after SUF treatment for 48 hours(p<0.01;p<0.001).The green fluorescence signal of OGFR was stronger.3.Using immunofluorescence and WB experiments,it was verified that shOGFR significantly reduced the expression of OGFR in H1975 and H23 cells(p<0.0001;p<0.001).The cloning experiment found that compared with the(sh NC+SUF)group,the number of H1975 and H23 cell clones in the(shOGFR+SUF)group increased,the clumps were enlarged,and the clone formation rates were 60.00%±7.22%(p<0.01)and54.93%±5.89%(p<0.01)respectively.Flow cytometry experiments found that,compared with the apoptosis rates of(sh NC+SUF)groups in H1975 and H23 cells(34.73% ± 2.40% and 29.29% ± 3.26%),ones of(shOGFR+SUF)groups in H1975 and H23 cells were siginificantly reduced(12.40%±1.31% and 13.58%±1.64%)(p<0.001;p<0.01).The results of WB experiments showed that compared with(sh NC+SUF)group,the expressions of Bax(p<0.01;p<0.0001)and cleaved caspaspe-3 of(shOGFR+SUF)groups in H1975 and H23 cells were significantly down-regulated(p<0.01;p<0.0001),and Bcl-2 protein up-regulated(p<0.01).Conclusion:1.The expressions of OGFR and MOR were diverse in different ADC cell lines.2.SUF can inhibit the proliferation,migration and invasion abilities of H1975 and H23 cell lines,and promote cell apoptosis.3.SUF can up-regulate the expression of OGFR and affect the growth of H1975 and H23 cell lines.