Research On The Function And Mechanism Of Long Noncoding RNA HITT In Tumor Drug Resistance

Cancer has become one of the major diseases threatening human health in the world.In China,cancer has surpassed cardiovascular diseases and become the disease with the highest mortality.Chemotherapy plays an important role in improving patient survival.Chemotherapy tolerance is one of major reasons for poor prognosis in tumor patients.Therefore,unveiling the molecular mechanism of chemotherapy resistance,exploring new directions and molecular targets for improving chemotherapy sensitivity are the hotspots in cancer research field s.Cells are unavoidably insulted by DNA damage.DDR(DNA damage response)is an important mechanism by which cells utilize to maintain genomic stability.Many clinical chemotherapy drugs kill tumor cells by causing cytotoxic DNA damage.Under such situations,the activation of DNA damage repair pathw ays inhibits cell death caused by chemotherapy drugs,and thus confers drug resistance.At present,targeted inhibition of the DNA repair signaling pathway has become an important strategy to enhance the sensitivity of cytotoxic treatments.Therefore,a better understanding of the regulatory mechanisms of DNA damage repair signalling is expected to provide new clues to improve treatment outcomes.ATM(ataxia-telangiectasia mutated),as a key regulator of the DNA damage response signaling pathway,its activation is mainly regulated by post-translational modifications.The activity of ATM controls a complex downstream signaling network like DNA repair,and plays important roles in determining the vulnerability to cell death.Reports have shown that inhibition of ATM kinase activity can improve the sensitivity of tumor cells to radiotherapy and chemotherapy.Revealing the regulatory mechanisms of ATM activity may provide important insights into ATM-targeted treatment.Recently,a lot of studies have demonstrated that long non-coding RNA(lnc RNA)plays an important role in regulating tumor drug resistance.Our previous work has identified an important lnc RNA HITT(HIF-1? inhibitor at translational level)that plays essential roles in inhibiting tumor growth by suppressing HIF-1?(hypoxia inducible factor-1?)expression.However,whether HITT is involved in the regulation of chemo-sensitivity has not been clarified.Thus,this thesis is aimed to explore the molecular mechanisms of HITT in regulating chemo-responses.Firstly,to identify if HITT play roles in DNA damage signaling pathway,the expression levels of HITT were analyzed after treating cells with different types of chemotherapeutic drugs.The results show that DNA damage-inducing drugs promoted the expression levels of HITT.And using Doxorubicin(Dox)as the representative DNA damage chemotherapy drug showed it induced HITT expression in dose-and time-dependent manners.Further mechanistic study revealed that Dox can increase the transcription level of HITT.And the transcription factor EGR1 contributes to the elevated HITT expression by binding at its promoter regions.Doxorubicin increased HITT expression by up-regulating EGR1 protein expression.These observation indicate that HITT may be involved in regulating DDR.Secondly,this thesis identify the regulation function of HITT with DNA damage agent Dox treatment,the experiment results showed that HITT plays a role in reducing DNA repair ability mediated by ATM kinase.Further molecular mechanistic study revealed that HITT directly bound with ATM,and chemotherapy drug Dox increased their binding by up-regulating HITT expression.And HITT through its third exon bind with ATM HEAT repeat domain,and ATM HEAT repeat domain is the key region mediated its binding with NBS1.As such,HITT blotted the interaction between ATM and NBS1,thus interfering the accumulation of ATM at the DNA broken sites and resulting in a reduced ATM activity.Thirdly,this thesis further explored the biological functions of HITT by regulating ATM activity.The results showed Dox induce cell apoptosis by increasing EGR1-HITT expression.In vitro cell experiments showed HITT enhance the sensitivity of different DNA damage chemotherapy drugs and this phenomenon were existed in different cell lines.Furthermore,the results derived from in vitro cell model and in vivo xenograft models all showed that HITT enhanced chemotherapeutic drug-induced killing effect by suppressing ATM activity.By analysing the colon cancer patient tissues,HITT was found to be down-regulated,while p-ATM is up-regulated in the tumor tissues in the comparison with the adjacent tissues,suggesting that the alerted p-ATM in tumor tissues may at least partially attribute to the reduced HITT expression.In summary,this thesis identifies a novel function of HITT in regulating DNA damage repair pathways.Mechanistically,it directly binds with ATM and inhib its MRN complex-mediated ATM recruitment and activation.By doing so,HITT expression successfully improves the sensitivity of tumor cells to chemotherapeutic drugs.These findings not only deepen our understanding of the mechanisms in regulating ATM activity,but also provide additional evidences on the tumor suppressive activities of HITT and important clues for ATM-targeted therapies to improve treatment outcomes.