Study Of The NETO2 Mediated Cellular Senescence In The Growth Of Prostate Cancer

PurposesIn the latest statistical data,prostate cancer is expected to rank the second in the world in terms of incidence rate and the fifth in terms of mortality,especially in the United States.The number of new cases of prostate cancer has exceeded that of lung cancer,ranking the first and the fifth in terms of mortality.In China,the incidence rate and mortality of prostate cancer have also gradually increased in recent years,which has posed a huge challenge to the medical system.The pathogenesis of prostate cancer is complex,but it is often accompanied by abnormal expression of certain genes.Cellular senescence is considered to be a stable and permanent state of cell cycle arrest,characterized mainly by inhibition of cell proliferation.NETO2 was first discovered to be expressed in brain tissue,and in the latest research,it has been found that it can promote the development of other cancers,including gastric cancer.In previous studies,our research team found that NETO2 has a close relationship with cellular senescence in prostate cancer,which may change the biological state of tumor cells through mediating cellular senescence.However,the relationship and role between NETO2 and cellular senescence are not very clear,which has aroused our interest in further research on it.In addition,drugs used to treat prostate cancer can also cause therapy induced senescence of tumor cells,and the relationship between this therapy induced senescence and NETO2 is also worth further study.This topic aims to explore the relationship between NETO2 and cellular senescence in prostate cancer,and further study the role of this connection in the growth of prostate cancer.MethodsUsing Enzalutamide to stimulate prostate cancer cells with gradient concentrations and different treatment times β-Galactosidase staining was used to detect the level of therapeutically induced senescence induced by it,in order to specifically describe the senescence pattern induced by Enzulutamide.In addition,prostate cancer cells were also treated with Enzalutamide at different concentrations and times as described above.Western blot experiments were used to detect the changes in NETO2 expression levels under their concentration gradients and different treatment times,and to verify the relationship between the senescence level induced by Enzalutamide and the changes in NETO2 level;Use 3D culture to further explore the relationship between therapy induced senescence induced by Enzalutamide and prostate cancer cell growth.Using lentivirus transfection to construct a stable NETO2 transfected prostate cancer cell line to observe whether actively changing NETO2 expression levels can cause changes in cellular senescence levels.At the same time,the growth status of various prostate cancer stable transfected strains under different senescence levels,as well as the growth trend and senescence level changes after treatment with Enzalutamide were observed.Finally,a NETO2 Nomogram was constructed using biological information methods to predict DFS in prostate cancer patients.ResultsBy observing the cellular senescence of three types of cells,LNCap,C4-2,and22RV1,stimulated by Enzalutamide at different concentration gradients and treatment times,we found that they were capable of producing a gradually increasing level of senescence,indicating that Enzalutamide could induce the senescence of LNCap,C4-2,and 22RV1,and that the degree of senescence was positively correlated with drug concentration and treatment time.Using the same concentration gradient and different duration of Enzalutamide treatment,we observed changes in the expression level of NETO2 in LNCap,C4-2,and 22RV1 cell lines.The protein expression level of NETO2 was negatively correlated with the drug concentration of Enzalutamide and the treatment time.Combining the positive correlation between Enzalutamide and cellular senescence,we preliminarily infer that the expression level of NETO2 may present a negative correlation with the level of cellular senescence.The 3D cell culture results showed that cellular senescence induced by Enzalutamide inhibited tumor growth,and the higher the degree of senescence,the more obvious the inhibition of tumor growth.The successful construction of stable transfection lines of NETO2 from various prostate cancer cell lines has led us to discover that actively changing the expression level of NETO2 can change the senescence level of prostate cancer cell lines LNCap,C4-2,22RV1,PC-3,and DU145.Furthermore,we have discovered that actively increasing the expression level of NETO2 can inhibit cellular senescence,while silencing NETO2 can improve cellular senescence level.Based on previous inference,We conclude that there is a negative correlation between the expression of NETO2 and cellular senescence.After actively changing the expression level of NETO2,AR expressing prostate cancer cell lines LNCap,C4-2,and 22RV1,as well as non AR expressing prostate cell lines PC-3 and DU145,all showed changes in senescence levels,indicating that NETO2 mediated changes in cellular senescence levels do not pass through the androgen receptor AR pathway.By observing the growth of various NETO2 stable transfection strains of prostate cancer cells,we found that NETO2 overexpression promotes the growth of prostate cancer cells by reducing the level of cell senescence,while NETO2 silencing inhibits the growth of prostate cancer cells by increasing the level of cellular senescence.After Enzalutamide treated each stable transfection strain,it was found that Enzalutamide further increased the senescence level of prostate cancer cell lines LNCap,C4-2,and22RV1 expressing androgen receptor AR,increasing its inhibitory effect on prostate cancer.However,changes in cellular senescence level and growth trend were not observed in prostate cancer cell lines PC-3 and DU 145 that did not express AR.Finally,a nomogram containing NETO2,clinical parameter T-stage,and Gleason score was constructed from the prostate cancer dataset in the tcga database to predict DFS for patients for 1,3,5 years.Conclusions1.Enzalutamide promotes prostate cancer cellular senescence in a time and concentration dependent manner and downregulates the expression of NETO2,and this therapy induced senescence caused by Enzalutamide acts as growth inhibition in prostate cancer cells.2.Overexpression/silencing of NETO2 results in changes in cellular senescence levels,indicating a negative correlation between NETO2 expression and cellular aging levels,and this change does not occur through the androgen receptor pathway.3.NETO2 mediated cellular senescence affects the growth of prostate cancer cells.Overexpression of NETO2 promotes prostate cancer cell growth by reducing cellular senescence levels,while silencing NETO2 inhibits prostate cancer cell growth by increasing cellular senescence levels.The AR(+)prostate cancer cell line can further inhibit its growth trend by increasing the level of cellular senescence after being treated with Enzalutamide.4.NETO2,clinical parameter T-staging,and Gleason score jointly predict DFS in prostate cancer patients.