Study On The Mechanism Of The Targeted Regulation Of RNUX3,miR-10b-5p And TIAM1 To Inhibit The Proliferation,Invasion And Migration Of Gastric Cancer Cells

Objective: To demonstrate the signaling pathways and relevant mechanism in the biological behaviors of proliferation,invasion and migration of gastric cancer cells,we investigated the regulatory relationships between RUNX3,mi R-10b-5p and TIAM1 at the tissue,cellular and molecular levels.Methods: 1.Prediction of micro RNAs: Through bioinformatics analysis,seven common online micro RNAs prediction databases,including Targetscan,mi RNet,mi RWalk,mi RDB,microt-cds,mi RSystem and mi RNAMap,were used to predict micro RNAs that might target TIAM1 m RNA 3'-UTR with comprehensive collection and coverage of current human micro RNA information,scientific and reasonable target gene prediction algorithm and extensive scientific research application.Eleven micro RNAs appearing in at least three databases were selected for experimental verification.2.To verify the targeting relationship between mi R-10b-5p and TIAM1:(1)Gastric cancer cell line BGC823 was transfected with mimics and inhibitors of 11 micro RNAs,respectively.After overexpression or inhibition of the corresponding micro RNAs,total RNA was extracted from the cells,and the changes of TIAM1 m RNA were detected by RT-q PCR after reverse transcription.(2)TIAM1 m RNA 3'-UTR full-length sequence was constructed into the 3'-end of the Renilla luciferase gene of psi-CHECKTM-2 vector,and the relationship between micro RNA and TIAM1 m RNA 3'-UTR was verified by double-luciferase assay in gastric cancer cell line BGC823.3.Expression of mi R-10b-5p in gastric cancer tissues and gastric cancer cell lines: The expression of mi R-10b-5p in 50 clinical tissue samples(including 25 cases of gastric cancer and 25 cases of corresponding adjacent 5cm normal tissue)was detected by RT-q PCR.The expression of mi R-10b-5p in 4 cell lines(including 3 gastric cancer cell lines BGC823,MGC803,SGC7901 and 1 normal gastric mucosal epithelial cell line GES-1)was detected 4.mi R-10b-5p inhibited gastric cancer cell proliferation,migration,colony formation and promoted apoptosis in gastric cancer cell line: BGC823 transfected with mi R-10b-5p-mimic or mi R-10b-5p-inhibitor,(1)through the CCK 8 test,we detected the OD450 nm absorbance value of BGC823 gastric cancer cells in 0 h,24 h,48 h,72 h.The more cells are the higher absorbance value is.Hence the OD450 nm values reflected the function of mi R-10b-5p on the proliferation of gastric cancer cells.(2)In Transwell test,we counted the BGC823 gastric cancer cells which migrated through the transwell membrane without or with matrix.The number of cells reflected the function of mi R-10b-5p on the ability of migration or invasion of gastric cancer cells.(3)In colony formation test,we counted the number of colony formation of gastric cells BGC823.The number of colony formation reflected the function of mi R-10b-5p on the ability of colony forming.(4)Flow cytometry was used to detect the percentage of early apoptotic cell population of gastric cancer cells BGC823 to observe the effect of mi R-10b-5p on the apoptosis of gastric cancer cells.5.Regulation of RUNX3 on mi R-10b-5p and TIAM1 expression:(1)The expression of RUNX3 was detected by RT-q PCR in 50 tissue samples and the correlation with mi R-10b-5p was analyzed.(2)The expression of mi R-10b-5p and TIAM1 was detected by RT-q PCR after transfecting RUNX3 expression vector(including RUNX3-1 and RUNX3-2)in gastric cancer cell line BGC823.(3)Construct RUNX3 gastric cancer-related single-base mutations(including RUNX3-1-R122 C and RUNX3-2-R136C)plasmids.To observe the effect of RUNX3 gastric cancer-related single-base mutations on the expression of mi R-10b-5p,we detected the expression of mi R-10b-5p after transfecting the mutant plasmids RUNX3-1-R122 C and RUNX3-2-R136 C.(4)To observe the effect of RUNX3 on Tiam1 protein,we used western blotting test after co-transfected RUNX3 expression vector and mi R-10b-5p-inhibitor.6.RUNX3 inhibits proliferation,invasion,colony formation and promotes apoptosis of gastric cancer cells: the effects of RUNX3 on proliferation,invasion,migration,colony formation and apoptosis of gastric cancer were observed by CCK-8 test,transwell chamber invasion and migration test,colony formation test and flow cytometry.7.CBF? assisted RUNX3 with regulating the expression of mi R-10b-5p:(1)Lentivirus transfection was used to construct gastric cancer cells with CBFB gene shutdown.(2)To observe whether RUNX3 could up-regulate the expression of mi R-10b-5p in the absence of CBF? in cells,we detected the expression of mi R-10b-5p after transfecting RUNX3 expression vector.(3)The expression of mi R-10b-5p was detected after co-transfection with RUNX3 and CBF? expression vector,and the upregulation of mi R-10b-5p by RUNX3 was observed when the intracellular CBF? was supplemented.8.CBF? assisted RUNX3 with inhibiting the proliferation,invasion and migration of gastric cancer cells:(1)After transfection with RUNX3 expression vector,CCK-8 assay and transwell chamber invasion and migration assay were used to observe whether RUNX3 could inhibit the proliferation,invasion and migration of gastric cancer cells in the absence of CBF?.(2)After co-transfection with RUNX3 and CBF? expression vectors,CCK-8 assay and transwell chamber invasion and migration assay were used to observe whether RUNX3 could restore the inhibitory effect on proliferation,migration and invasion of gastric cancer cells when intracellular CBF? was supplemented.9.The effect of CBF? and RUNX3 on the possible promoter regions of the precursor sequence of mi R-10b-5p: the possible promoter regions of the precursor sequence of mi R-10b-5p(-2268~-170)were amplified from the genomic DNA of the normal gastric mucosal epithelial cell line GES-1.Four truncated sequences of different lengths were amplified(P-pro A:-2268~-170,P-pro B:-1764~-170,P-pro C:-1266~-170,P-pro D:-1266~-170).The 5'-end of Firefly Luciferase gene was constructed on the p GL3-basic vector.(1)In gastric cancer cell line BGC823,we detected the luciferase activity after transfecting the plasmids.The luciferase activity indicated the promoter activity of the amplified fragments.(2)To observe the effect of RUNX3 on the promoter activity,we used the dual-luciferase assay to detecte the changes of luciferase activity after co-transfecting the plasmids and RUNX3 expression vector.The changes in luciferase activity indirectly reflected the changes in promoter activity.(3)To observe the effect of RUNX3 on promoter activity of truncated fragment in the presence of CBF?,we detected the luciferase activity after co-transfecting fragment plasmids,RUNX3 expression vector and CBF? expression vector in CBFB shutdown cells.Results: 1.mi R-10b-5p directly regulates the expression of TIAM1 m RNA 3'-UTR(1)Bioinformatics method predicted and selected 11 micro RNAs targeting TIAM1 m RNA 3'-UTR.Eight of them(including mi R-10b-5p,mi R-589-3p,mi R-651-3p,mi R-653-5p,mi R-373-3p,mi R-372-3p and mi R-205-3p)influence on the expression of TIAM1 m RNA at different degrees,with statistical significance.Mi R-4770,mi R-592 and mi R-7844-5p did not affect the expression of TIAM1 m RNA.(2)Dual-luciferase assay showed that only mi R-10b-5p could affect the 3'-UTR of TIAM1 m RNA among the above 8 micro RNAs that had an effect on the expression of TIAM1 m RNA,which was statistically significant.2.mi R-10b-5p is down-expressed in gastric cancer tissues and cell lines and inhibits gastric cancer proliferation,invasion and migration,colony formation and promotes apoptosis.(1)The expression of mi R-10b-5p in gastric cancer tissues was lower than that in the normal tissues 5cm away from the cancer,with statistical significance(p=0.0064).And the expression in gastric cancer cell lines BGC823,MGC803 and SGC7901 was lower than that in human normal gastric mucosal epithelial cell lines GES-1,with statistical significance.(2)After transfection with mi R-10b-5p-mimic in gastric cancer cell line BGC823,the absorbance value of gastric cancer cells at 450 nm after 0h,24 h,48h and 72 h was significantly lower than that of the control group.After transfection with mi R-10b-5p-inhibitor,the absorbance of gastric cancer cells at 450 nm increased significantly at 0h,24 h,48h and 72 h after transfection with mi R-10b-5p-inhibitor.3.RUNX3 regulates the expression of mi R-10b-5p and TIAM1 and inhibits the proliferation,invasion and migration,colony formation and apoptosis of gastric cancer(1)The expression of RUNX3 in gastric cancer tissues was lower than that in the normal tissues 5cm away from the cancer,and was positively correlated with the expression of mi R-10b-5p(r=0.3725),with a statistically significant correlation(p=0.0231).(2)After transfection with RUNX3 expression vector in gastric cancer cell line BGC823,RUNX3-1 and RUNX3-2 both increase the expression of mi R-10b-5p by about 3-4 times,with statistical significance.After transfection with RUNX3-1-R122 C,the expression of mi R-10b-5p in gastric cancer cell line BGC823 decreased to the same level as that in the control group,with statistical significance.After transfection with RUNX3-2-R136 C,the expression of mi R-10b-5p only was with decreasing trend.(3)After transfection with RUNX3 in gastric cancer cell line BGC823,the absorbance value of gastric cancer cells at 450 nm at 0h,24 h,48h and 72 h was significantly lower than that of the control group.The number of cells migrating through the transwell chamber was significantly reduced,the number of colony formation was significantly reduced,and the percentage of apoptotic cell group was increased.(4)After transfection with RUNX3 expression vector in gastric cancer cell line BGC823,RUNX3-1 reduced the expression of TIAM1 m RNA by about 2 times in the two subtypes of RUNX3,with statistical significance.RUNX3-2 showed only a decreasing trend for TIAM1 m RNA.After co-transfection of RUNX3-1 and mi R-10b-5p-inhibitor,RUNX3-1 lost its inhibitory effect on Tiam1 protein expression.4.CBF? assisted RUNX3 with upregulating the expression of mi R-10b-5p and inhibiting proliferation,invasion and migration of gastric cancer(1)In the stable CBFB-shutdown cells,the expression of mi R-10b-5p was about 2 times lower compared with that in the common gastric cancer cells with statistical significance.In the stable CBFB-shutdown cells,the expression of mi R-10b-5p in the RUNX3-1 group(which transfected RUNX3-1 alone)was not increased compared with control group.The expression of mi R-10b-5p in the CBF?/RUNX3-1group(which co-transfected RUNX3-1 and CBFB)was about 2 times higher compared with the control group with statistical significance.Similarly,in the stable CBFB-shutdown cells,the expression of mi R-10b-5p in the RUNX3-2 group(which transfected RUNX3-2 alone)was about 1.5 times higher compared with the control group with statistical significance.The expression of mi R-10b-5p in the CBF?/RUNX3-2 group(which co-transfected RUNX3-2 and CBFB)was about 2.5 times higher compared with the control group with statistical significance.Co-transfection of RUNX3 and CBFB had a stronger effect on upregulation of mi R-10b-5p expression than transfection of RUNX3 alone.(2)In the stable CBFB-shutdown cells,the relative OD450 values and the numbers of migrated and invaded cells did not differ in the RUNX3-1 group compared with the control group.After co-transfecting RUNX3-1 and CBFB,the relative OD450 values and the numbers of migrating and invaded cells were significantly lower than in the control group.Similarly,in the stable CBFB-shutdown cells,the relative OD450 values and the numbers of migrated and invaded cells in the RUNX3-2 group were still significantly lower than in the control group and those in the co-transfected group showed the lowest values among all three groups(control group,RUNX3-2 group,co-transfected RUNX3-2,and CBFB group).(3)p GL3 plasmids containing four truncated fragments of mi R-10b-5p precursor sequence upstream(P-pro A:-2268~-170,P-pro B:-1764~-170,P-pro C:-1266~-170,P-pro D:-732~-170)showed luciferase expression in gastric cancer cell BGC823.Among the four fragments,p GL3-P-pro A showed the highest luciferase activity and was most affected by RUNX3.After co-transfecting RUNX3,CBF? and p GL3-P-pro A,the luciferase activity was higher than that after co-transfecting RUNX3 and p GL3-P-pro A.Conclusion:(1)The expression of RUNX3 presented decreased trend in gastric cancer tissues and was positively correlated with the expression of mi R-10b-5p.The expression of mi R-10b-5p was reduced in gastric cancer tissues and cell lines.The expression of TIAM1 was increased in gastric cancer cell lines.RUNX3,mi R-10b-5p and TIAM1 were correlated in expression.(2)CBF? assisted RUNX3 with up-regulating the expression of mi R-10b-5p.Mi R-10b-5p inhibited the expression of TIAM1 by directly targeting m RNA-3 'UTR.After the inhibition of mi R-10b-5p,RUNX3 lost its inhibitory effect on Tiam1 protein expression.It suggests that RUNX3 could down-regulate the expression of TIAM1 by up-regulating mi R-10b-5p.(3)CBF? assisted RUNX3 with inhibiting the proliferation,invasion and migration of gastric cancer cells;RUNX3 inhibited the formation of gastric cancer colonies and promoted apoptosis;mi R-10b-5p inhibited the proliferation,invasion and migration of gastric cancer,colony formation and promoted apoptosis.CBF?/RUNX3 may play a biological role by upregulating mi R-10b-5p.(4)This study demonstrated for the first time that CBF? assisted RUNX3 with inhibiting the expression of TIAM1 by up-regulating mi R-10b-5p,thereby inhibiting the proliferation,invasion and migration of gastric cancer cells.