The Establishment Of A Radioresistant Hepatocellular Carcinoma Cell Line And The Preliminary Study Of Preliminary Study Of Radioresistance Mechanism

Background:Hepatocellular carcinoma(HCC)is the fifth most common solid malignant tumor and consided to be the second leading cause of cancer-related deaths worldwide,of which the occourence has been increasing rapidly in the last 25 years according to the newest statics report relased in recent years[1].As for the HCC patients,most of which are not available for surgion on account of large tumor size,multifocality vascular invasion,resulting in severe limitation in the expansion of hepatic transplantation for patients with HCC[2].Radiotherapy,regarded as one of the most important method for HCC therapy.Dispite that many patients got huge benifit from radiation technology,locoregional recurrence or metastatic spread still occurs in response to radioresistance.As a result,we should plunge right into specific and efficient research about radioresistance which may lead to advances in the progress of HCCIt has been widely reported that radioresistant heterogeneous populations,as one part of cancer cells,were responsible for radioresistance of tumor.But according to the most research nowadays,there are rarely acceptable methods by which several cancer cell lines with acquired radioresistant characteristics and behaviours have been established.For minimizing the time and money,there is no time left for us to grope a way to bulid a stable and reliable radioresistant cell platform of hepatocellular carcinoma for the further studies.According to previous data and report,we have the concept that activity of the DNA damage pathway could induce many materails like Y-H2AX,ATM,ATR,which were acceptablely known as the early and reliable surrogate for DNA double-strand breaks(DSBs)and were once used to detcet how the DSBs produce and repair(Banath et al.,2010)[3].Late S and G2/M phases of the cell cycle is when homologous recombination(HR)which is one of major pathways in repairing DSBS that plays an essential role in maintaining genome integrity,which requires a sister chromatid as a template(Chemikova et al.,2012 an Mladenov et al.2013)[4,5].Caspase 3,BCL-2,BAX are believed to be greatly related genes of aposis[6].Epcam,one factor that is regarded to be the biomarker of stermness of hepatocellular carcinoma,which can activate WNT signal pathway and induce ehemoresistence[7].At the same time,it can deaden the abilitiy of aposis.We tend to further understand the underlying mechanism of the radioresistance in MHCC-97H-R cell line,we firstly sought to measure the biological characteristics including property of DNA damage repair,rate of aposis,cell cycle,eell stermness and so on to hold on some possible clues for its radioresistance·SLUG,a member of the Snail family of highly evolutionarily conserved zinc-finger transcription factors,which is detectable in many types of cancer[8].Previous studies have demonstrated that SLUG overexpression exhibits a radioprotective function in TK6 cells demonstrating that it played as a part in gene therapeutic radioprotection of normal tissuesp[9].More than that,sLUG can induce EMT and contribute to the stermness of cancer cells SLUG is implicated in many signaling pathways,including radiation-induced apoptosis in hematopoietic stem and progenitor cells[10].Transcriptional regulation and post-transcriptional regulation are widely eccepted that they play the most important parts of gene expression in the complex regulation network.LNCRNAs,as a class of small molecules of noncoding single-stranded RNA,is widely exist in eukaryotes and also play an important role in post-transcriptional regulation in many kinds of tumor occourence and development.But its role in tumor radiation resistance is still unclear.Therefore,the LNCRNAs choosen from of a microarray(radioresistant cell line vsus parental cells)may provide new targets and theoretical basis for HCC radiosensitizationThis study aims to construct a stable and reliable radioresistant cell platform of hepatocellular carcinoma and use gene chip technology to find possible LNCRNA to explore the mechanisms of radioresistance.Objestive1.Establishing the Hepatocellular carcinoma radioresistant cell paltform(MHCC-97H-R)for the investigation of hepatocellular carcinoma radioresistant mechanisms.2.Detecting the biological character of MHCC-97H-R including propensity of stem,apoptotic,proliferation and DNA double-strand repair capacity.3.Investigating that SLUG plays a part in the regulation of hepatocellular carcinoma radiation resistance through affecting tumor stemness.4.Providing LNCRNAs to investigate the possible mechanism in the regulation of MHCC-97H-R cell line radioresistance.Material and Methods:1.Establishing the hepatocellular carcinoma radioresistant cell lines with conventional dose fractionation mode(exposured to radiation(2GY/F)for 15times,20 times,25 times,30 times)and sublethal dose mode(exposured to radiation(2Gy/F,4Gy/F,6Gy/F,8Gy/F,10Gy/F).2.Colony formation assay and muti-target and one-hit model were used for analysis of radioresistance.3.Cell apoptosis and cell cycle,and expression of epcam were detected by using flow cytometry.The CCK-8 method was used to measure cell proliferation.Comet assay and Edu incorporation assay were used to determine the cell DNA damage repair capacity.Real-time PCR and Western-blot was used to detect the expression of DNA damage response and apoptosis related genes(Y-H2AX and Caspase 3,BCL-2,BAX),and EMT related genes.4.Detecting the expression of SLUG,stemness related genes(OCT-4,NANOG,SOX-2),DNA double-strand repair capacity related genes(CHK1,ATM,CHK2,ATR,Y-H2AX)in different times after exposuring to radiation(6GY/F)5.The LNCRNA microarray about hepatocellular carcinoma radioresistant cell line(MHCC-97H-R)and MHCC-97H cell line(both exposured to 6Gy of 6MVX-ray radiation after 1 hour)was done by shanghai kangcheng company.Results:1.The 2Gyx25times group MHCC-97H-R cell lines behaves the most strongest radiation resistantance(mean lethal dose DO=2.612Gy,While the contronled cell line's is 1.434Gy).We named it MHCC-97H-R.The sublethal dose for the investigation of Hepatocellular carcinoma is 6Gy.2.After 24h exposed to 6Gy of 6MVX-ray radiation,lower apoptotic,higher proliferation rate appeared in MHCC-97H-R(14.47%to 25.21%,P<0.01))than that of parental cells,and it emerges a significant cell cycle G2 arrest(63.04%to 49.82%,P<0.01)).The comet assay showed significantly shortened comet tail of MHCC-97H-R than that of parental cell line.After lh exposed to 6Gy of 6MVX-ray radiation,Western blot indicates that Y-H2AX is lower expression in MHCC-97H-R and 5-ethyl-2'-deoxy uridine(EdU)incorporation experiment shows that MHCC-97H-R cell lines took more EdU which is a DNA repair material mixed analogue.The expression of Epacm is idealy higher expression in MHCC-97H-R.3.By successfully knocking down the expression of slug,the expression of cell stemness related genes NANOG,OCT-4,SOX-2 KIF-4decreases,and the expression of ATM-Chk2 decreases and the expression of ATR-Chkl increases in 12h,24h After 6Gy of 6MVX-ray radiation,which may indicate that SLUG plays a part in regulation of radioresistance through cell stemness.4.The results of the LNCRNA microarray:in 695 differentially expressed LNCRNA of MHCC-97H-R and MHCC-97H cell line(Fold Change?2?P-value?0.05),313with increased expression LNCRNAs and 214 with decreased.Among the increased difference expression LNCRNAs,176 of them are intronic antisense or natural antisense with its nearby coding gene and the coordinate of the coding gene which are more advised to be investigated,while 84 of decreased are choosen.In the GO and Pathway analysis,comparing wiht parental cancer cell,there are 8 increased-expressed LNCRNAs involved in Cytosolic DNA-sensing pathway as well as 9 involved in TNF signaling pathway and 29 involved in apoptotic deaths·Conclusion:The hepatocellular carcinoma radioresistant cell lines(2Gyx25times group,named MHCC-97H-R)were established by colony formation assay with conventional dose fractionation mode,and analysisd by muti-target and one-hit model,which exhibits lower apoptotic propensity,stronger ability of DNA repair and proliferation,higher expression of Epcam and significant G2 phase arrest when exposed to 6Gy of 6MVX-ray radiation.The LNCRNA microarray also provide suffficent LNCRNAs which are Fold Change?2,P-value?0.05 in statistics may be key roles in radioresistance.