| BackgroundLung cancer,as one of the most common malignant tumor,which cause serious damage to physical and mental health of the people around the world and bring an indelible disaster to the patient and his family.With the continuous development of science and technology,and medical advances,human gradually had already overcome many of the diseases.But the tumor has been difficult problems to solve.And most of the people's health consciousness is weak.When they feel very uncomfortable,the discovery is the terminal cancer.So they missed the best time to treatment,which brought heavy pain to the patient's family,so it is very urgent to the prevention and treatment of lung cancer.At present,there are four main types of lung cancer treatment basic methods:surgical treatment,radiotherapy,chemotherapy and targeted therapy.Although surgery and radiotherapy had made some progress,but the prognosis of lung cancer is very poor.The five-year survival rate of lung cancer in china is only 16.1%.Lung cancer mainly includes two types,respectively is Non-Small Cell Lung Cancer and Small Cell Lung Cancer.Small cell lung cancer accounts for the proportion of about 20%in the lung cancer.The doubling time in small cell lung cancer is short.And the lung cancer had high proliferation rate and wide transfer early.Surgery is only suitable for small resectable patients with stage I non-small-cell lung cancer,about 2%to 5%.The currently diagnosed with stage I non-small-cell lung cancer is not more than 5%of the overall small cell lung cancer patients,and the patients beyond the T1-2N0 cannot benefit from surgery.In patients with bureau within a time limit,the effective rate of etoposide plus cisplatin chemotherapy combined chest after radiotherapy is 70%-90%.And in patients with extensive period,the expected effective rate of simple combination chemotherapy is 60%-70%.Although small cell lung cancer is highly sensitive to initial chemotherapy and radiotherapy,the malignant degree is very high and most of the patients died of recurrence in the end.The median survial for patients with bureau deadline is 14 to 20 months,the median survial for patients with extensive period is 9 to 11 months.After reasonable treatment,the survival rate in bureau deadline patients is about 40%in two years,extensive period of two years survival rate less than 5%.High incidence and low survival rate after reasonable treatment are the prevention and control of the situation in the present stage small cell lung cancer.So research on the treatment of small cell lung cancer and has been the focus of scientific research workers at home and abroad.Serine/threonine kinase33(STK33)is a member of serine/threonine kinase(STK)superfamily.The phosphorylated proteins play an important role in cell proliferation,differentiation,apoptosis and tumor metastasis.STK33 gene on chromosome 11,11 p15.3 area,and includes 12 introns and 1545 bp open reading frame.STK33 often highly expressed in different kinds of tumors,especially in lung adenocarcinoma and large cell lung cancer.Ribosomal S6 kinase 1(ribosome protein subunit 6 kinase 1,S6K1)is rapamycin target protein(mammalian target ofrapamycin,mTOR)of a downstream molecules.S6K1 activation can phosphorylate the ribosomal protein S6(RPS6),thus further promote cell proliferation differentiation and related protein synthesis.BAD is a member of the family of the Bcl-2,as is known to all members of the family of the Bcl-2 mainly includes three members.one member can inhibit the cell apoptosis including Bcl-2,BHRF1,Bcl-X1 and Ced-9.Another member can enhance cell apoptosis including BAD,Bax,Bak,Bcl-Xs;finally a gene is involved in the mediation cells survive.So the proteins S6K1,RPS6 and BAD could regulate cell apoptosis and invasion.It is predicted that S6K1/RPS6/BAD signal pathway may be the mechanism of STK33 gene regulating small cell lung cancer,but need to be further in-depth discussion.AimsThe aim of this study is to explore whether STK33 regulate the proliferation,invasion,and apoptosis of small cell lung cancer through regulating S6K1/RPS6/BAD signal pathway.Clarify the mechanism of STK33 inhibition small cell lung cancer through regulating S6K1/RPS6/BAD signal pathway.These can provide more theoretical basis for better clinical testing and clinical treatment and medical workers.MethodsThis study choose pGCsi-H1 with shRNA carrier construction of plasmid STK33 interference,transfection into classic NCI-small cell lung cancer cell line H446 in the method,using Polymerase Chain Reaction lock Real-time Polymerase Chain Reaction(RT-qPCR)and protein immunoblot(western blot,WB)technology to detect different shRNA interference efficiency.We detect small cell lung cancer cell viability by MTT test.Further,we detect cell cycle and apoptosis of small cell lung cancer cell proliferation and apoptosis by FCM(flow cytometry).We detect the effect of STK33 to small cell lung cancer cells about invasion and migration ability in vitro.Using normal NCI H446 cells,steady turn pRNA H1.1 plasmid of NCI H446 cells,steady turn pRNA H1.1-shSTK33 plasmid NCI-H446 cells respectively to construct nude mice transplanted tumor model,observe the nude mice transplantation tumor growth,further reflect the STK33 on small cell lung cancer cells in vivo tumor generation capability.The transplanted tumor tissue by immunohistochemical technique(immunohistochemical analysis,IHC)analysis STK33 p-S6K1 in the organization,the influence of p-BAD protein expression.After the RT-PCR technology to detect lower STK33 RPS6,S6K1,caspase 9,the expression of BAD changes.WB technology further defined STK33 RPS6,p-RPS6,S6K1,p-S6K1,cleaved caspase 9,BAD,p-BAD expression,to further clarify its role.Results1.Knock-down STK33 reduced the proliferation of small cell lung cancer cells According to MTT experiment results,we found that knock-down STK33 can restrain small cell lung cancer cell survival,the experiment result can be in the time series of experiments.To different types of cells incubated with the common 120 hours,every 24 hours,with a determined by MTT colorimetric method to determine record OD490 values in each group.Cell viability in shSTK33 group were lower than the control group,and NC group of each point in time,and with other shSTK33 group differences between the two groups have statistical significance(P<0.05),suggesting that the STK33 played important roles in small cell lung cancer cell proliferation.2.Knock-down STK33 could induce small cell lung cancer cell apoptosis and stop the G1 cell cycle.We found that the knock-down STK33 can induce small cell lung cancer cell apoptosis by flow cytometry results.Cells apoptosis rate in the control group on average respectively:(NCI-H446 cells apoptosis rate of 3.7±0.5%,DMS153 cells 2.8±0.3%).The NC group of average cell apoptosis rate is respectively:(NCI-4.2±0.6%H446 cells,cells DMS153 3.7± 0.6%).The average shSTK33 group of cells apoptosis rate is respectively:(NCI-12.7± 0.6%H446 cells,cells DMS153 15.6:±1.1%).The statistical results show that shSTK33 group with other differences between the two groups were significant(P<0.05)).Furthermore,knock-down STK33 also prevent small cell lung cancer cell NCI-H446 G1 cell cycle.In shSTK33 group,cells distributed in G1 phase average higher than the control group,and NC group,shSTK33 group with other differences between the two groups were significant(P<0.05).Comprehensive the above results determined by MTT method and shows that STK33 in small cell lung cancer cell survival and played an important role in survival rate,inhibit the occurrence and development of STK33 can significantly inhibit tumor,thereby to tumorigenesis.3.Knock-down STK33 inhibited the invasion ability small cell lung cancer cellsThe degree of malignancy of tumor depends on the invasive ability of tumor cells.Therefore,we use Transwell invasion assay and Wound healing assay to assess the effect of knock-down STK33 on invasion in small cell lung cancer cell.Transwell assay results showed that compared with the control group,and NC group,small cell lung cancer cell group after knock-down STK33,through the polycarbonate membrane cells was reduced.In order to further understand the STK33 for small cell lung cancer cell migration ability of inhibiting effect,through the cell scratch experiment results,we found that the same STK33 shRNA cells than without carrying the gene wound closure to delay,scratches the distance to be more big.To sum up,STK33 not only afects the survival and proliferation of small cell lung cancer cells and the potential of the tumor plays a decisive role,knock-down STK33 can significantly inhibit the small cell lung cancer cell invasion and migration ability.4.Knock-down STK33 played an important role in small cell lung cancer by inhibiting RPS6/BAD signaling pathwaysIn order to further validate the role of RPS6/BAD signal pathway in small cell lung cancer cells,the STK33 regulating by Western and PCR technique to detect S6K1 RPS6/BAD signal path expression.The way has been proved related to the function of STK33 in non-small cell lung cancer.However,in the current study,lower STK33 has no effect on S6K1 activities.On the pathway of other indicators expressing condition is the same with previous report.Found that knock-down STK33 reduced RPS6 phosphorylation,S6K1 downstream effector need ribosomes and protein synthesis.Knock-down STK33 also inhibits the expression of p-BAD,but has no effect on total BAD.In addition,knock-down STK33 induced cracking cysteine protease 9,suggesting that suppressed STK33 induction of apoptosis is mediated by mitochondrial pathway.We inhibited the role of STK33 in cancer by using ML281.By specific inhibitors inhibit STK33 cell survival rate at the same time reduce the nci-h446 cells showed a similar effect,on similar to the effect of shRNA RPS6/BAD.We could conduct a conclusion:STK33 inhibition the apoptosis is mediated by mitochondrial pathway.5.Knock-down STK33 limits the small cell lung cancer cell growth,and can suppress RPS6/BAD signal through a small cell lung cancer xenograft model ratsIn vitro experimental results further were tested in animal models.We will made from different types of cells suspension and,by means of subcutaneous immunization vaccination to rat skin.And in seven days after transplantation tumor growth.We observe the tumor volume increased,solid tumor can be detected until 22 days.And on the 16th day,observed nude mice transplanted tumors had accumulated shSTK33 group was obviously lower than the control group or NC group(P<0.05).The transplanted tumor tissue,extraction of histones,WB found that shSTK33 RPS6 p and p-BAD expression levels were significantly lower than control group or NC group(p<0.05),and the expression of cracking cysteine protease in shSTK33 group 9 is significantly higher than the control group,and NC group,the results with the results of in vitro experiments are consistent.To sum up,the results show that STK33 gene in the development of small cell lung cancer has played a key role.Conclusions:1.Knock-down STK33 SCLC cells could significantly inhibit cell proliferation and invasion,and induce cell apoptosis at the same time.2.That is associated with phenotypic characteristics change,knock-down STK33 also decreased RPS6 phosphorylation and BAD(apoptosis related proteins)and increase the expression of cracking cysteine protease 9,suggests that knock-down STK33 induced cell apoptosis by mitochondrial pathway mediation.3.We also found that STK33 inhibitors also can significantly inhibit cell activity of small cell lung cancer cells,and can significantly inhibit RPS6/BAD signaling pathway.4.In addition,the knock-down STK33 also can restrain the growth of the transplanted tumor and the expression of RPS6/BAD.We found STK33 down-expression induced apoptosis by mitochondria in SCLC cell,so we speculated that STK33 may have specific characteristics of cancer.Meaning:We clarified the mechanism of STK33 inhibition small cell lung cancer by regulating S6K1/RPS6/BAD signaling pathway for the first time.These results provide more theoretical basis for better clinical testing and clinical treatment and more evidence for medical workers. |
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