Studies On The Effect And Mechanism Of LncRNA TMPOP2 On DNA Damage Repair Of Human Cervical Cancer Cells

Cervical cancer(CC)is a malignant tumor caused by human papillomavirus(HPV)infection of the female reproductive system.Radiation therapy and chemotherapy are the main methods of traditional therapies of cervical cancer.The principle of radiotherapy and certain chemotherapy drugs for cervical cancer is related to causing a large amount of DNA damage in cells.Therefore,exploring molecular targets that are related to DNA damage may provide new ideas for improving cervical cancer treatment.Long noncoding RNA(LncRNA)refers to a kind of RNA molecules that are longer than 200 nucleotides.LncRNAs have no protein-coding function and play a role in many biological activities.LncRNA TMPOP2 is a recently identified LncRNA highly expressed in cervical cancer cells.In order to study the functions of TMPOP2,we used siRNA to knock down the expression of TMPOP2 in HeLa cells,then performed transcriptome sequencing to identify genes that were affected by TMPOP2.Various life-related processes including cell cycle,cell invasion and metastasis,DNA damage repair were revealed to be affected by TMPOP2.This project mainly focused on the relationship between TMPOP2 and DNA damage repair.First,TMPOP2 was knocked down in HeLa cells to detect DNA damage repair related genes-RFWD3/DDB1/RAD21/TMPO.Western blot and RT-qPCR results showed that the expression of RFWD3/DDB1/RAD21/TMPO decreased in TMPOP2-depleting cells.In contrast,RFWD3/DDB1/RAD21/TMPO expression was increased when TMPOP2 was overexpressed in HeLa cells.Similar results were observed in HPV16-positive CaSki cells,demonstrating that TMPOP2 affects the expression of genes related to DNA damage repair in cervical cancer cells.In order to further explore the effect of TMPOP2 on DNA damage repair,first,DNA damage marker yH2A.X was detected by Western blot and immunofluorescence experiments.Knockdown of TMPOP2 in HeLa cells using siRNA showed that the level of yH2A.X protein was increased.Subsequently,comet assays were used to detect DNA damage.The results showed that tailing cells increased significantly and the tailing length became longer in the TMPOP2-knockdown group.In addition,repeated verifications on HPV 16-positive CaSki cells also gave consistent results.The above results indicate that knocking down of TMPOP2 causes DNA damage in cells.Finally,CaSki cells overexpressing TMPOP2 were treated with cisplatin,a DNA damage inducer.Compared with the control group,the protein level of yH2A.X decreased faster with the increase of repair time,suggesting that TMPOP2 promotes DNA damage repair.Previous studies have shown that HPV E6/E7 and TMPOP2 promote each other’s expression,and HPV E6/E7 also affects DNA damage repair.In order to identify whether the promotion effect of TMPOP2 on DNA damage repair is mediated by HPV E6/E7,this project further examined the effect of HPV E6/E7 on DNA damage repair related genes.Western blot results show that HPV E6/E7 and TMPOP2 have opposite effects,so TMPOP2 affects DNA damage repair differently from HPV E6/E7.Subsequently,experiments were performed with HPV-negative colon cancer HT-29 cells which were demonstrated to highly express TMPOP2 as well,and the results showed that TMPOP2 still affects the expression of DNA damage repair-related genes,proving that the relationship between TMPOP2 and DNA damage repair is independent of HPV E6/E7.In order to further test whether TMPOP2 affects the sensitivity of cervical cancer cells to the chemotherapeutic drug cisplatin,cervical cancer cells were treated with siTMPOP2 in combination with cisplatin,and apoptosis was detected by flow cytometry and Western blot experiments.The results showed that knocking down TMPOP2 can increase the sensitivity of cervical cancer cells to cisplatin by inducing more cells to undergo apoptosis.In this study,a series of experiments at the molecular and cellular levels revealed the relationship between LncRNA TMPOP2 and DNA damage repair for the first time:TMPOP2 affects DNA damage repair-related gene expression and promotes DNA repair.Knockdown of TMPOP2 enhances the sensitivity of cervical cancer cells to chemotherapy,suggesting that TMPOP2 is a potential target for cervical cancer treatment.