The Role Of Long Noncoding RNA-HOTTIP In Renal Cell Cancer And Resistance To Targeted Drugs

Renal cell carcinoma(RCC)is among the most common forms of malignancies originating from the human kidneys,which accounts for 2%to 3%of all adult malignancies and causes 100,000 deaths per year worldwide.Owing to the help of abdominal imaging,most RCC patients diagnosed at the early stages can be surgically cured.While about 25%of patients are diagnosed with locally advanced or metastatic disease at first,furthermore,20-40%of patients with local tumor who undergo surgical resection,will develop metastatic RCC subsequently.Individuals with advanced disease face poor prognosis.Agents targeting angiogenesis have played a primary role in the treatment of metastatic renal cell carcinoma.However,resistance to anti-angiogenesis therapy almost always occurs which makes RCC still a refractory disease worldwide.Therefore,it is essential for improvement of RCC therapy to further understand the molecular mechanism of RCC and find effective targets to predict disease metastasis and drug resistance.LncRNAs are a class of RNAs constituted of more than 200 nucleotides in length and playing significant roles in gene transcription,translation,post-transcriptional control and posttranslational modification.Given that many IncRNAs are expressed in the way of tissue and cancer type restriction,specifc lncRNAs are now likely to be translated into clinical applications for diagnosis,prognosis or predicting the response of treatmentRecently;aggregating evidence has demonstrated the aberrant expressions of various lncRNAs are associated with the tumorigenesis,development and progression of RCC.The HOXA transcript at the distal tip(HOTTIP)lncRNA,located at the 5’ end of the HOXA cluster,was recently functionally characterized.Increased HOTTIP expression has been reported in tongue squamous cell carcinoma,lung cancer,pancreatic cancer,hepatocellular carcinoma,osteosarcoma,gastric cancer and so on.In these tumors,HOTTIP may serve as a potential oncogene and be an adverse prognostic factor in patients.However,little is known about the role of HOTTIP expression in the tumorigenesis and development of RCC.The aim of this study was to investigate role of HOTTIP in RCC and find out the specific mechanism of HOTTIP affecting RCC biological behavior and resistance to sunitinib.’ Through the study of this subject,we expect to find new molecular targets in the development of RCC to better predict the prognosis of RCC,guide the selection of targeted drugs and explore the solutions after drug resistance.Part Ⅰ The expression level and role of HOTTIP in renal cell cancer Objective:To explore the expression level of HOTTIP in renal cell cancer and its correlation with clinicopathological parameters and prognosis of patients,moreover,to investigate the effect of HOTTIP on renal cell cancer cell proliferation,cell cycle,migration,invasion and tumourigenesis in nude mice.Methods:Real-time quantitative PCR(qRT-PCR)was used to detect the expression of HOTTIP in renal cell cancer tissues and paired normal tissues.The relationship between the expression of HOTTIP and clinicopathological parameters and prognosis of renal cell cancer was analyzed-Stable transfection was done with Letivirus to knock-down or enhance HOTTIP expression in Caki-1 and Caki-2 cell lines.The influence of HOTTIP on cell function was evaluated by CCK-8 assay,colony formation assay,flow cytometry and transwell invasion migration experiments in vitro.In addition,xenograft studies using nude mice in vivo were performed.Results:qRT-PCR results showed that HOTTIP was significantly overexpressed in renal cell lines and tissues.Compared with patients with low expression of HOTTIP,patients with high expression of HOTTIP had higher grade of tumor stage,higher incidence of distant metastasis,and lower overall survival rate.Additionally,cell function test in Caki-1 and Caki-2 indicated that cell proliferation,migration and invasion abilities in HOTTIP over-expressed group were enhanced compared with the negative control(NC)group,while HOTTIP knockdown showed the opposite results.Subcutaneous xenograft tumor model displayed that knockdown of HOTTIP could impede tumorigenesis in vivo.Conclusion:HOTTIP was highly expressed in renal cell cancer and might be used as a novel prognostic indicator for renal cell cancer.HOTTIP could promote proliferation,migration and invasion of renal cell cancer cells in vitro and in vivo.Part Ⅱ Effects of HOTTIP on the biological function and the resistance to sunitinib in renal cell cancer through regulating c-Met pathwayObjective:To explore the specific mechanism of HOTTIP influencing the cell function and resistance to sunitinib of renal cell cancer.Methods:Stable transfection was done with Letivirus to knock-down or enhance HOTTIP expression in Caki-1 and Caki-2 cell lines.Western blot was used to detect the expression level of key molecules on c-Met pathway.Cell proliferation,invasion and metastasis abilities were evaluated in HOTTIP overexpression cells after treating with the c-Met inhibitor SU11274.CCK8 was used to detect the cell activities under different concentration of sunitinib with or without previously treating with the c-Met inhibitor SU11274 in HOTTIP overexpression or knockdown cells.Results:The expression levels of c-Met,p-c-Met,p-PI3K and p-AKT in HOTTIP knockdown cell lines were significantly lower than those in control group while the above protein expressions were obviously increased in HOTTIP overexpression group.The abilities of cell proliferation,clonal formation and invasion and migration were significantly weakened after adding the c-Met inhibitor SU11274 in HOTTIP overexpression cell lines.In the drug resistance assays,knockdown of HOTTIP decreased the resistance to sunitinib while HOTTIP overexpression had an opposite effect.There was no significant difference in the resistance to sunitinib between HOTTIP overexpression and knockdown gruops adding SU11274.Conclusion:HOTTIP might play an important role in the cell proliferation,invasion and migration of renal cell cancer by regulating c-Met pathway.In addition,HOTTIP promoted the drug resistance to sunitinib through activating c-Met pathway and the resistance could be reversed by c-Met inhibitor SU11274.