Effect Of ART1 On IL-6-induced Proliferation Of Colon Cancer Cells Through Regulating Gp130

Research background and purpose:Colorectal cancer(CRC)is the fourth most common cancer in the world with the second highest mortality rate,and the fourth most common cancer in China.Due to many factors involved in the carcinogenesis and development process,it has been difficult to make breakthroughs in the prevention and treatment of CRC.It is estimated that the global burden of CRC will increase by 60% in the next 10 years.Chronic inflammation is now recognized as a risk factor for CRC,it can induce carcinogenesis and promote the development of CRC.Among the many cancer-promoting inflammatory factors,IL-6 plays a central role.However,the clinical anti-IL-6 monoclonal antibody has no obvious effect on CRC.Therefore,to explore the relevant regulatory factors of IL-6-induced proliferation of colon cancer cells in CRC is helpful to find new therapeutic targets.Arginine-specific mono-ADP-ribosyltransferases-1(ART1)is a positive regulator of inflammatory cytokines,and inhibition of ART1 in alveolar type II epithelial cells can inhibit the release of IL-6.Our previous studies have shown that ART1 is closely related to various biological behaviors of CRC.More importantly,ART1 knockdown can reduce the phosphorylation level of the promoter of the signal transduction molecule gp130 in IL-6 signaling pathway.ART1 has not been reported on the effect and mechanism of cancer cell growth induced by IL-6 in colorectal cancer.Therefore,this subject intends to explore whether ART1 can affect IL-6-induced proliferation of colorectal cancer CT26 cells through regulating gp130 and the possible mechanisms,so as to provide an experimental basis for finding new potential targets for CRC treatment.Method: 1.Expression of ART1 and gp130 in colorectal cancer The expressions of ART1 and gp130 in 54 cancer cases of colorectal cancer and 23 control tissues were detected by immunohistochemistry.The correlation between ART1 and gp130 expression was analyzed.2.The effect of ART1 knockdown on the IL-6-induced proliferation of colon cancer CT26 cells Three groups of CT26 cells were selected: ART1 knockdown group(ART1-sh group),negative control group(NC group),un-transfected group(WT group),of which WT and NC groups were control groups,ART1-sh group was the experimental group.(1)IL-6 induced proliferation of colorectal cancer CT26 cells 1)CCK-8 was used to detect the cell proliferation activity of colon cancer CT26 cells induced by different concentrations of IL-6 for different times.2)Western blot was used to detect protein levels of c-myc,CyclinD1 and Bcl-xl induced by IL-6 in colon cancer CT26 cells at different times.(2)The effect of ART1 knockdown on IL-6-induced proliferation of CT26 colon cancer cells 1)CCK-8 was used to detect the cell proliferation activity induced by IL-6 in three groups of colon cancer CT26 cells.2)Western blot was used to detect protein levels of c-myc,CyclinD1 and Bcl-xl in three groups of colon cancer CT26 cells induced by IL-6.3)Plate colony formation experiment and Edu experiment were used to detect the colony formation and DNA synthesis of three groups of colon cancer CT26 cells induced by IL-6.(3)Colitis mice model were established by drinking DSS solution.NC and ART1-sh group CT26 cells were transplanted subcutaneously in the flank of colitis mice,and the effect of ART1 knockdown on the growth of transplanted tumor induced by IL-6 was observed.3.The mechanism of ART1 knockdown on IL-6-induced proliferation of colorectal cancer CT26 cells Three groups of CT26 cells were selected: ART1 knockdown group(ART1-sh group),negative control group(NC group),un-transfected group(WT group),of which WT and NC groups were control groups,ART1-sh group was the experimental group.(1)Western blot was used to detect protein levels of gp130 and p-STAT3 in three groups of colon cancer CT26 cells with or without IL-6-induction.(2)Q-PCR was used to detect the mRNA levels of gp130 and IL-6 in three groups of colon cancer CT26 cells.(3)The dual-label immunofluorescence was used to initially detect the spatial relationship between ART1 and gp130 in colon cancer CT26 cells.(4)Western blot was used to detect protein level of gp130 and p-STAT3 in transplanted tumor tissues of NC and ART1-sh group.Result: 1.Expression of ART1 and gp130 in colorectal cancer The positive rates of ART1 and gp130 expression in human CRC tissue samples were higher than those in control tissues,and the positive rates of the two were 90.7% and 83.3%,respectively.There is a positive correlation between the expression of these two proteins in cancer tissues(P<0.05).2.Effect of ART1 knockdown on the proliferation of colon cancer CT26 cells induced by IL-6(1)IL-6 induces proliferation of colon cancer CT26 cells 1)After incubating colon cancer CT26 cells with different concentrations of IL-6 for different times,the proliferation activity of the cells increased with a time-and concentration-dependent manner within a certain range(P<0.05).2)The IL-6-induced protein level of c-myc,CyclinD1 and Bcl-xl in colon cancer CT26 cells increased with the induction time(P<0.05).(2)The effect of ART1 knockdown on IL-6-induced proliferation of CT26 colon cancer cells 1)Three groups of colon cancer CT26 cells were incubated with different concentrations of IL-6.IL-6-induced proliferation activity of ART1 knockdown group was significantly lower than that in the control group(P<0.05).2)The rate of IL-6-induced colony formation in ART1 knockdown group was significantly lower than that in the control group(P<0.05).3)IL-6-induced DNA synthesis in ART1 knockdown group was significantly lower than that in the control group(P<0.05).4)IL-6-induced expressions of c-myc,CyclinD1 and Bcl-xl in the ART1 knockdown group were significantly lower than those in the control group(P<0.05).(3)After successfully establishing a colitis mouse model,colon cancer CT26 cells of NC and ART1-sh groups were transplanted subcutaneously in flank region of the mice,and the transplanted tumors will form 2 weeks later.The volume of transplanted tumor of ART1 knockdown group was significantly smaller than that in NC group(P<0.05).(4)The protein levels of c-myc,CyclinD1 and Bcl-xl in transplanted tumors of ART1 knockdown group were lower than those of the NC group(P<0.05).3.The mechanism of ART1 knockdown on IL-6-induced proliferation of colon cancer CT26 cells(1)In three groups of colon cancer CT26 cells without IL-6 incubation,the protein level of gp130 and p-STAT3 in the ART1 knockdown group was lower than that in the control group(P<0.05).(2)In three groups of colon cancer CT26 cells without IL-6 induction,the levels of gp130 mRNA and IL-6 mRNA in the ART1 knockdown group were lower than those in the control group(P<0.05).(3)The IL-6-induced protein levels of gp130 and p-STAT3 in colon cancer CT26 cells gradually increased with a time-and concentration-dependent manner within a certain degree(P<0.05).(4)The IL-6-induced protein levels of gp130 and p-STAT3 in ART1 knockdown group were lower than those in the control group(<0.05).(5)Colon cancer CT26 cells were simultaneously staining with ART1 and gp130,and the co-localization of the two was analyzed in ImageJ.The results show that the two have a moderately strong correlation.(6)The protein levels of gp130 and p-STAT3 in transplanted tumors of ART1 knockdown group in colitis mice were lower than those in NC group(P<0.05).Conclusion:The positive rates of ART1 and gp130 expression in human CRC tissue were higher than those in the control group,and there was a positive correlation between these two proteins.ART1 promotes the proliferation of colon cancer cells induced by IL-6,which may be related to the upregulation of p-STA3 by increasing the expression of IL-6 signal transducer gp130,and then promotes the expression of c-myc,CyclinD1 and Bcl-xl.The detailed mechanisms need further study.