| Objective:Long non-coding RNAs(LncRNA)are involved in almost all stages of cancer development,and their expression shows more tissue-and cell-specific patterns than protein-coding genes,so they are considered as potential biomarkers and therapeutic targets in cancer.In addition,a growing number of studies have demonstrated that LncRNA regulates the expression and function of zeste homologue 2(EZH2)at multiple levels,thereby regulating the tumorigenesis process.Recent studies have shown that LncRNA SNHG1 is highly expressed in Cervical Cancer(CC)tissues compared with normal tissues;however,the mechanism of action of SNHG1 in CC cells remains unclear.In this study,a cervical cancer SiHa cell model with low expression of SNHG1 and EZH2 and low expression of SNHG1 and overexpression of EZH2 was constructed to investigate the effects of SNHG1 and EZH2 on the proliferation,invasion and apoptosis of SiHa cells.Methods:In this study,plasmid and siRNA were transfected into SiHa cells.The cells were divided into five groups: siRNA NC(NC),SNHG1 siRNA(si-SNHG1),EZH2 siRNA(siEZH2),SNHG1 siRNA + blank vector(si-SNHG1 + con),si-SNHG1 + EZH2 overexpression vector(si-SNHG1 + EZH2)was used to detect the changes of cell growth ability after transfection by CCK8 method.transwell assay was used to observe the changes of cell invasion ability after transfection.Flow cytometry was used to detect the changes of cell proliferation cycle and apoptosis after transfection.RNA immunoprecipitation(RIP)was performed in SiHa cells with anti-EZH2 and anti-igg antibodies to understand the interaction between SNHG1 and EZH2.All statistical analyses were performed by SPSS 26.0 and Graphpad Prism 8.0 software,and measurement data were expressed as mean ± standard deviation(±s).Independent sample t test was used for comparison between the two groups,and one-way analysis of variance was used to compare the differences between multiple groups.All experiments were repeated at least three times,with P<0.05 indicating statistically significant differences.Results:1.The OD value of cells in each group at 450 nm for 24 h,48h and 72 h was detected by CCK8 assay.At 24 h,compared with NC group(0.60±0.02),the cell proliferation capacity of si-SNHG1(0.33±0.04)and si-EZH2 group(0.29±0.04)was significantly decreased(P < 0.001),compared with si-SNHG1 + con group(0.36±0.06).si-SNHG1 +EZH2 group(0.50±0.04)reversed the inhibitory effect of SNHG1 knockdown on cervical cancer cell proliferation(P < 0.05);At 48 h,compared with NC group(1.32±0.07),the cell proliferation capacity of si-SNHG1(0.53±0.10)and si-EZH2 group(0.43±0.06)was significantly decreased(P < 0.001),compared with si-SNHG1 + con group(0.64±0.14).siSNHG1 + EZH2 group(1.19±0.05)reversed the inhibitory effect of SNHG1 knockdown on cervical cancer cell proliferation(P < 0.01);At 72 h,compared with NC group(2.36±0.23),the cell proliferation capacity of si-SNHG1(1.14±0.13)and si-EZH2 group(1.39±0.16)was significantly decreased(P < 0.001),compared with si-SNHG1 + con group(1.46±0.22).si-SNHG1 + EZH2 group(2.25±0.11)reversed the inhibitory effect of SNHG1 knockdown on cervical cancer cell proliferation(P < 0.01);2.Cell cycle changes in each group were analyzed by flow cytometry: Compared with NC group(49.87±0.86),the proportion of cells in G0/G1 phase in si-SNHG1 group(68.12±1.12)and si-EZH2 group(66.40±3.34)was increased(P < 0.01).Compared with si-SNHG1 + EZH2 group(55.54±1.65),si-SNHG1 + EZH2 group(64.64±2.92)decreased(P < 0.01);On the contrary,compared with NC group(45.06±1.38),the proportion of cells in S phase in si-SNHG1(19.05±0.02)and si-EZH2 group(24.97±0.22)decreased(P <0.01).(55.54±1.65)in si-SNHG1 + EZH2 group was higher than that in si-SNHG1 + con group(64.64±2.92)(P < 0.01);3.Flow cytometry analysis of cell apoptosis ratio(%): Compared with NC group(5.47±1.02),si-SNHG1(12.30±2.03),si-EZH2 group(11.85±0.30)significantly increased apoptosis rate(P < 0.001);Compared with si-SNHG1 + con group(12.53±0.80),siSNHG1 + EZH2 group(8.83±1.61)reversed the ability of SNHG1 knockdown to promote apoptosis of cervical cancer cells(P < 0.05);4.Transwell assay was used to detect the effect of low expression of SNHG1 and EZH2 on the invasion ability of SiHa cells: Compared with NC group(2306.00±99.23),the number of downward invasion of cervical cancer cells in si-SNHG1 group(797.00±40.29)and si-EZH2 group(875.33±73.53)was significantly decreased(P <0.001).Knockdown of SNHG1 and EZH2 could effectively inhibit the distant metastasis of cervical cancer cells.Compared with si-SNHG1 + con group(889.33±82.03),si-SNHG1 +EZH2 group(1482.00±141.74)reversed the inhibition ability of SNHG1 knockdown on cervical cancer cell invasion(P < 0.01).Conclusion:This study showed that lncRNA-SNHG1 targeting EZH2 gene is involved in regulating the proliferation,invasion and apoptosis of cervical cancer SiHa cells,providing new ideas and potential therapeutic targets for the improvement of cervical cancer. |
