Rapamycin May Inhibit Murine S180 Sarcoma Growth By Regulating The Pathways Associated With Autophagy And Cancer Stem Cells

The doctoral dissertation is composed of two parts.Part I Rapamycin may Inhibit Murine S180 Sarcoma Growth by Regulating the Pathways Associated with Autophagy and Cancer Stem CellsBackground:With the continuous development of modern society,the annual incidence rate of cancer in Chinese is increasing,and the mortality rate is increasing year by year.China is a vast country with a huge population,accounting for about 20%of the world's total population.In the 21st century,the proportion of birth rate and mortality has gradually increased.In the world environment,nearly 22%of new cancer patients and 27%of patients who died of cancer come from China every year.The corresponding cost of cancer treatment is increasing year by year,which has seriously restricted the development and progress of society.Social pressure,environmental pollution,diet mode tend to westernization,exercise reduction and aging population are the main reasons for the increase of incidence rate.The concept of sarcoma is a group of tumors with special biological manifestations originated from mesenchymal components.It is malignant but different from the cancer we often say.Most of the sarcomas are located in epidermis,subcutaneous,bone,soft tissue,connective tissue and so on.There are also sarcomas in the interstitium of the lung Sarcoma(PPS)which is a rare sarcoma of the respiratory system,accounting for 0.7%?3.6%of the malignant tumors in China.According to the origin of the diseased tissues,sarcomas can be divided into leiomyosarcoma,liposarcoma,fibrosarcoma,malignant mesothelioma,etc.Although the incidence of sarcoma is low,it can occur at any age.The number of male patients is higher than that of women.Compared with common cancers such as lung cancer and colorectal cancer,the incidence rate of cancer tends to be low and age is higher,and the proportion of young people and middle-aged people is higher.The prognosis is not good.Sarcomas are not sensitive to radiotherapy and chemotherapy.The adjuvant treatment before and after surgery has no obvious advantage in extending patient survival time.The surgical intervention is still a relatively effective way.The purpose of surgery is to try our best to achieve complete resection of the tumor,which has a great impact on the prognosis.Therefore,targeted therapy is a more scientific and targeted treatment for sarcomas.The main gene mutations of sarcomas include KRAS,PIK3CA,EGFR,etc.the targeted drugs for KRAS mutation and EGFR mutation are MEK inhibitors and Tiki drugs respectively.MTOR inhibitor is a kind of drug targeting at the change of signal pathway of PIK3CA gene.MTOR(mammalia target of rapamycin)is a mammalian target of rapamycin,which has two complex forms.Also known as mTORCl and mtorc2,the mechanism of mTOR signaling pathway is to participate in the process of tumor occurrence and inhibit the apoptosis of tumor cells.In addition,mTOR is an endogenous inhibitor of autophagy.Apoptosis also occurs in tumor tissue,which is mediated by autophagy.At present,many kinds of mTOR inhibitors have been developed,and their targets involve various pathways of mTOR signals.Rapamycin(rapa)plays a role in mTORC1 by binding to intracellular receptors,but has no significant effect on mtorc2.Rapamycin can inhibit the activation of growth factors such as IL1,IL2 and colony stimulating factors,and the activation of oncogenes due to the over activation of PI3K and Akt.At the same time,rapamycin can also affect the production of peripheral blood vessels,cut off free channels and limit the spread of tumor cells by reducing the material sources of supply cells.In the study of melanoma,rhabdomyosarcoma,pancreatic cancer,leukemia and other tumor cells,the antitumor effect has been shown.Rapamycin blocked the progress of mitotic cycle,could not complete the copy process,and has been proved to promote the apoptosis process.Objective:At present,there are few studies on rapamycin in sarcoma,and its function and mechanism of action are not fully clear.Therefore,it is of great significance to study the inhibition function and mechanism of rapamycin in sarcoma.In this study,the target signaling pathway was associated with autophagy and tumor stem cells,and the mechanism of rapamycin effect on the growth of sarcoma was discussed in depth,so as to provide experimental basis for the drug adaptability of sarcoma treatment.Methods:In this study,S180 sarcoma cells were cultured and the effect of rapamycin on S180 cell apoptosis was detected by flow cytometry and CCK8 test.The effects of rapamycin on the formation,invasion and migration of S180 cells were detected by Transwell test.In animal experiments,S180 sarcoma of mice was used as the research object to establish the tumor bearing model of S180 sarcoma cells of mice.The mice were treated with rapamycin at different concentrations.After the experiment,the mice were killed and the tumor tissues were separated.The histopathological changes of S180 sarcoma were observed by HE staining and the expression and localization of mTOR,Beclin1,LC3,ulkl,CD 133,CD90 and Notch1 proteins in S180 sarcoma were detected by laser confocal microscopy.The autophagy related proteins mTOR,Beclin 1,LC3(micro associated protein light chain 3),ulkl and tumor stem cell markers CD 133,CD90,Notch 1(UNC-51 like autophagy activating kinase)were detected by real-time PCR and Western blot,respectively 1)To explore the mechanism of rapamycin on 5180 sarcoma from animal level and cell molecular level.Result:1.Rapamycin induced S180 sarcoma cell apoptosis in a dose-dependent mannerThe results of flow cytometry showed that the apoptosis of S180 cells increased with the increase of rapamycin concentration for 24 hours,and with the increase of rapamycin concentration,the number of early and late apoptosis cells increased significantly.It is suggested that rapamycin can induce apoptosis of S180 cells in a dose-dependent manner.2.Rapamycin inhibited the migration and invasion of S180 sarcoma cells.After rapamycin treatment,the migration ability of S180 cells decreased significantly,and there was significant difference between the high concentration rapamycin group and the control group.The results of Transwell showed that the invasion ability of S180 cells decreased after rapamycin treatment,and there was significant difference between the high concentration rapamycin group and the control group.All these results suggested that rapamycin inhibited the ability of clone formation,migration and invasion of S180 cells in a dose-dependent manner.3.Rapamycin inhibited the growth of S180 sarcoma in tumor bearing mice.The average volume of subcutaneous tumor in mice treated with rapamycin was lower than that in the control group.Rapamycin(2 mg/kg)inhibited tumor growth,but there was no significant difference between the two groups.In the rapamycin(4 mg/kg)treatment group,tumor growth was significantly inhibited.On the 14th day,the average sarcoma volume of the untreated group was 2298 mm3,while that of the rapamycin treated group was 1264 mm3.The tumor growth inhibition rate of rapamycin 2 mg/kg and rapamycin 4 mg/kg was 30.1%and 48.8%,respectively.These results suggest that the effect of rapamycin on the growth of S180 sarcoma is related to the dosage of rapamycin.4.Rapamycin can promote the apoptosis of S180 sarcoma cells,and the degree of apoptosis is affected by the dosage of drugs.After he staining,the cells of S180 sarcoma tissue of tumor bearing mice showed irregular growth,and the arrangement order was lamellar and nested.In particular,the shape,color and size of the core are different,and the thickness of the nuclear membrane is not uniform.With the increase of the concentration of rapamycin,the proportion of cell necrosis,vacuole and nuclear pyknosis increased significantly.5.Rapamycin inhibited the expression of mTOR and promoted the expression of Beclinl,ulkl and LC3 in S180 sarcoma cells.The expression of mTOR in 4 mg/kg rapamycin group was significantly different from that in the control group(P<0.05).In S180 sarcoma cells,Beclinl and LC3 were expressed in cytoplasm and cell membrane,ulkl and mTOR were mainly expressed in cytoplasm.In the cultured cells and tumor tissues,real-time PCR and Western blot showed that compared with the control group,the mRNA and protein expression of ulkl,Beclin 1 and LC3 increased after rapamycin treatment,and the expression of ulkl and Beclin 1 in the 4 mg/kg rapamycin group was significantly different from that in the control group(P<0.05),suggesting that rapamycin can promote the transcription and expression of ulk1 and Beclin 1.The expression of LC3 II/I protein was significantly up-regulated in the high-dose and low-dose rapamycin groups(P<0.05).6.Rapamycin inhibited the expression of CD90,CD 133 and Notch 1 protein in S180 sarcoma cells.Immunohistochemistry and immunofluorescence showed that Notch 1,CD 133 and CD90 were expressed in cytoplasm and cell membrane of S180 sarcoma cells.The results of real-time PCR and Western blot showed that the mRNA and protein expression of Notch 1,CD 133 and CD90 in S180 sarcoma cells were down regulated after different concentrations of rapamycin compared with the control group,and the expression of Notch 1,CD 133 and CD90 in 4 mg/kg rapamycin group was significantly different from that in the control group(P<0.05).It is suggested that rapamycin can inhibit the transcription and expression of CD90,CD 133 and Notch 1 in S180 sarcoma in a dose-dependent manner.Conclusion:Rapamycin can inhibit the growth of S180 sarcoma cells in a dose-dependent manner,kill the tumor and induce the apoptosis of S180 sarcoma cells.Rapamycin inhibited the migration and invasion of S180 sarcoma cells.Rapamycin can promote the transcription and expression of Beclinl,ulkl and LC3 in S180 sarcoma cells,and inhibit the transcription and expression of mTOR,CD90,CD 133 and Notch 1 in a dose-dependent manner.It is suggested that the inhibition of rapamycin on the growth of S180 sarcoma and the induction of apoptosis may be related to its promotion of autophagy and inhibition of the expression of tumor stem cell markers.Part ? The mechanism of LACTB regulating EMT in inhibiting metastasis and invasion of lung adenocarcinomaResearch significance:Lung cancer is one of the most malignant tumors in the world with the highest morbidity and mortality.The incidence rate of lung cancer is one of the highest malignant tumors in the world.The incidence rate of lung adenocarcinoma is about 25%of the primary lung cancer.The incidence of lung cancer is relatively small.Compared with squamous cell carcinoma of lung,the incidence of lung cancer is higher and easy to metastases.Patients with lung adenocarcinoma are not sufficiently sensitive to current radiotherapy and chemotherapy during treatment,and their prognosis is poor.The 5-year overall survival rate of patients is very low,less than 15%.Therefore,finding new treatment targets and providing patients with more reasonable treatment plans will have a positive effect on the treatment of cancer and have important significance for achieving accurate treatment of lung adenocarcinoma.Research purposes and methods:Recent studies have shown that a mitochondrial protein lactamase,LACTB,has been reported to have cancer suppressing functions,but its function and mechanism of action have not been reported in lung cancer.EMT of lung cancer is closely related to metastasis and recurrence.The purpose of this study is to explore the function and mechanism of LACTB in regulating EMT,metastasis and invasion in lung adenocarcinoma.We used TCGA data to analyze the expression of lactb mRNA in lung adenocarcinoma and the level of promoter methylation,and analyzed the correlation between the expression of lactb and the survival of patients.The expression of lactb in lung adenocarcinoma cells was detected,and then the effect on cell proliferation and migration was detected by means of MTT,scratch test,Transwell and so on.The effect on cell EMT was detected,the expression of MMP2 and EMT markers and the change of related signal molecules were detected,so as to explore the effect of lactb on EMT and transformation of lung adenocarcinoma cells The regulation mechanism of migration and invasion.Experimental results:1.LACTB mRNA is under-expressed in lung adenocarcinoma,and promoter methylation is increased.LACTB expression is positively correlated with patient survival.Patients with low-LACTB expression have significantly less survival than patients with high-expression.2.LACTB overexpression inhibits EMT in lung adenocarcinoma by down-regulating Snail,E-cadherin expression is up-regulated,N-cadherin and Vimentin expression are down-regulated;Snail expression is up-regulated after interfering with LACTB expression,and promotes EMT in lung adenocarcinoma.3.LACTB overexpression down-regulates the expression of MMP2 to inhibit the metastasis and invasion of lung adenocarcinoma cells;after interfering with the expression of LACTB,MMP2 expression is up-regulated to promote the metastasis and invasion of lung adenocarcinoma cells.Experimental results:1.LACTB is underexpressed in lung adenocarcinoma and positively correlated with patient survival;2.LACTB inhibits EMT by down-regulating Snail,thereby inhibiting metastasis and invasion of lung adenocarcinoma.