Mechanism Of LncRNA H19 Regulating Radiosensitivity Of Colon Adenocarcinoma Cells

Based on the global cancer data forecast,the death cases of colorectal cancer were935,000 in 2020,accounting for about 9.4 percent of the global cancer death cases.Colorectal cancer had emerged as the second most deadly malignancy in the world.Radiotherapy is one of the treatments for colorectal cancer,which has the advantages of narrowing tumor size and reducing distant metastasis.However,radioresistance of tumor is an important cause of radiotherapy failure for colorectal cancer.Studies have identified that long non-coding RNA H19(lncRNA H19,H19)acts the oncogene with poor prognosis in colorectal cancer,and H19 participates with the biological processes including proliferation,epithelial mesenchymal transition,autophagy,chemoresistance,invasion and migration of colorectal cancer cells through various mechanisms such as miRNA sponge and RNA-binding protein.However,it remains unclear whether H19 is involved in the regulation of radiosensitivity in colorectal cancer until now.In this study,we studied the regulation of H19 on the radiosensitivity of colon adenocarcinoma cells after X-ray radiation and its mechanism,aiming to provide a potential target for prognosis evaluation and improvement of radiotherapy efficacy of colorectal cancer.Firstly,bioinformatics methods were applied to obtain the expression of H19 in colon adenocarcinoma and clinical prognosis data using TCGA,ln CAR and other databases,and the relevance was analyzed.Then,RT-q PCR was performed to detect the expression of H19 in three colon adenocarcinoma cell lines after X-ray radiation,and clonal formation assay was used to compare the radiosensitivity of different H19 expression cell lines.The influences of H19 silencing and overexpression on the proliferation,DNA replication and radiosensitivity of colon adenocarcinoma cells were researched by CCK-8,Ed U and clonal formation assay.Bioinformatics predicted the downstream miRNA of H19,and RT-q PCR screening and double luciferase assay was adopted to verify whether H19 combined with miR-193b-3p,while CCK8,Ed U and clonal formation assay were operated to detect the regulation of miR-193b-3p on the radiosensitivity of colon adenocarcinoma cancer cells.Subsequently,a series of molecular biological approaches were used to predict,screen and finally verify whether MDM2 was the downstream m RNA of miR-193b-3p,and the effect of MDM2 on the radiosensitivity of colon adenocarcinoma cancer cells was investigated.Flow cytometry analysis and Western blot were performed to examine the influences of H19,miR-193b-3p and MDM2 on cell cycle distribution after X-ray radiation.Finally,rescue experiment was conducted to verify the accuracy of H19/miR-193b-3p/ MDM2 signaling axis.The research results showed that:(1)The expression of H19 in colon adenocarcinoma tissues was higher than that in normal colon epithelial tissues,and the expression of H19 was significantly correlated with clinicopathology and TNM stage.Additionally,the high expression of H19 was positively associated with the poor prognosis of colon adenocarcinoma patients.So,H19 could be used as one of the prognostic indicators of colon adenocarcinoma.(2)The expression of H19 were upregulated in all three cell lines after X-ray radiation,and the high expression of H19 was positively correlated with the radioresistance of colon adenocarcinoma cells.(3)Silencing of H19 in HCT116 and HT29 cells inhibited cell proliferation,DNA synthesis and clone formation after irradiation.In contrast,overexpression of H19 promoted these effects in HT29 and SW620 cells.Meanwhile,cell cycle analysis showed that the combination of silencing H19 and radiation promoted G2/M phase arrest.(4)There was a negative correlation between miR-193b-3p expression and H19 in colon adenocarcinoma cells,indicating a direct combination between the two.Upregulation of miR-193b-3p had an inhibitory effect on cell proliferation,DNA synthesis and clonal formation in colon adenocarcinoma cells.In addition,upregulation of miR-193b-3p could shift the radiation-induced G2/M arrest to G0/G1 phase.(5)Upregulation of miR-193b-3p could inhibit the expression of MDM2,while silencing of H19 also suppressed it.Moreover,miR-193b-3p and MDM2 had a binding interaction.Knockdown of MDM2 could alleviate cell proliferation,DNA synthesis and clonal formation,and silencing MDM2 moved the radiation-induced G2/M arrest to G0/G1 phase.(6)Upregulation of miR-193b-3p reversed the promotion of cell proliferation by overexpression of H19.Similarly,silencing MDM2 relieved the proliferationpromoting effect of overexpressed H19 and inhibited cell proliferation.These results indicated that H19 enhanced the radioresistance of colon adenocarcinoma cells to X-ray through affecting the cell cycle distribution by the miR-193b-3p/MDM2 signaling axis,which suggested that H19 was expected to be a potential target for radiation sensitization and a biomarker for prognosis assessment of colorectal cancer.