Translated Abstract
Background:
As the increasing morbidity and mortality globally, cancer has been the main reason of morbidity and significant public health issue of different popluation in China. According to the report of National Cancer Center, the number of new cases and death cases accounts for 21.8% and 27% all over the world, which means that China is ranking above the average in the pool of 184 countries. The morbidity of renal cell carcinoma(RCC) in Chinese is 66.8%(with male accounting for 43.2%, while female accounting for 23.6%, the rate is 1.83:1 ); on the other hand the mortality is 23.4%( with male accounting for 15.2%, while female accounting for 8.2%, the rate is 1.85:1). The statistic of American cancer demonstrates that renal cell carcinoma is top ten malignant tumor of both male and female in the US, which accounts for 3.7% of new cases, estimating the number of new cases in 2017 is 639990. A larger account of patients suffering from RCC were detected via imageological examination by chance. Although the RCC detected in primary stage is usually with good prognosis, 25 percent patients have been developed into later period meaning metastasis and poor prognosis. The uptodate research about the mechanism of RCC progression and breakthrough of target therapy in the past ten years redefinite RCC, especially the treatment of metastatic RCC. However, with the augment of drug resistance, innovative mechinism of RCC progression and target for drugs are calling for in-deapth research. Long noncoding RNA is a type of RNA which have no protein coding ability. It can interact with a certain protein and be involved into modulation of gene transcription and epigenetic modification, functioning as activator, decoy, inducer and scaffold; while in the level of post-transcription, lncRNA can give rise to mRNA, splice mRNA, or could be spliced into short ncRNA. As the first indentified lncRNA in eukaryote, lncRNA HOTAIR has unique high level expression in multiple tumors functioning as antisense modulator. Given the recent researches, lncRNA HOTAIR is powerful, playing a big oncogenetic role in several kinds of cancers. But we still have little knowledge about the function of lncRNA HOTAIR in the progression of RCC. Although lncRNA HOTAIR owns many ability to promote cancer development, the existing report about HOTAIR and RCC are focusing on proliferation and metatasis with epigenetic modulation, lacking researches in the direction of growth suppression, motility, immortality, and angiogenesis. Vascularization is the markable sign of RCC. Angiogenesis gives rise to proliferation, invasion and metastasis of RCC and the suppression of angiogenesis is the target of targeted therapy of RCC, particularly the advanced renal cell carcinoma. Based on the discoveries of basic and clinical research, we found out that the illumination of mechinism of lncRNA HOTAIR and progression of RCC is insufficient, and the report about the lncRNA HOTAIR and angiogenis is rare.
Objective:
Based on TCGA database and research in vitro, this program elemently discusses the relationship between lncRNA HOTAIR and RCC; By reconstructing cell model in vitro, this program explores the molecular mechanisms of lncRNA HOTAIR promoting vascularization in RCC and explicits whether lncRNA HOTAIR can be a new target of “vessel targeted therapy” or a new sufficient marker of “vessel targeted therapy” of RCC.
Methods:
1. We cited the graphs of lncRNAtor datebase, TANRIC database and CCLE datebase to demonstrate the expression level of lncRNA HOTAIR in different cancer tissues, the relationship of expression level of lncRNA HOTAIR andsurvival time and the difference of expression level of lncRNA HOTAIR in various types of RCC;Seperating data from TCGA database into normal and primary tumor,, we analysed the discrepancy of transcriptional level of lncRNA HOTAIR、VHL、HIF-2α and VEGFa using t-test to detect the statistic differences;
2. The transcription level of lncRNA HOTAIR in normal renal tubular epithelial cell and five kinds of renal cell carcinoma were dectected by real-time qPCR.The expression level of HIF-2 α and AR in normal renal tubular epithelial cell and five kinds of renal cell carcinoma were dectected by real-time qPCR and Western Blot;
3. Using siRNA to knockdown lncRNA HOTAIR in OSRC-2 and SW839 cell models; Using pcDNA3.1(+) overexpression vector to construct lncRNA HOTAIR-overexpressed OSRC-2 cell model; Using lentivirus transfection system to knockdown AR in SW839 cell model.
4. The influence of OSRC-2 to HUVEC after knockdowning and overexpressing lncRNA HOTAIR were detected by Transwell assay and tube formation assay; The influence of SW839 to HUVEC after knockdowning lncRNA HOTAIR and AR were detected by Transwell assay and tube formation assay;
5. The transcription level of lncRNA HOTAIR and the expression level of AR、HIF-2α and VEGFa were examined by real-time qPCR in OSRC-2/Vector、OSRC-2/HOTAIR、OSRC-2/siNC、OSRC-2/siHOTAIR、SW839/shNC、 SW839/shAR、SW839/siNC、SW839/siHOTAIR.
Results:
1. The expression level of lncRNA HOTAIR in clear cell renal cell carcinoma and papillary renal cell carcinoma are significantly higher than normal tissues; the higher the expression of lncRNA HOTAIR shows the lower the survival probability; the expression levle of lncRNA HOTAIR is highest in cell lines belong to clear cell renal cell carcinoma, rather than other pathological classification; the genes associated with vascularization have significantly higher expression level in primary tumor than in normal tissue.
2. The transcription level of lncRNA HOTAIR of SW839, 786-O, OSRC-2, ACHN and 769-P are verious, and the highest is SW839; while the highest transcription level of HIF-2α is ACHN; the expression of protein HIF-2α can only be detected in HK-2(weakly positve), 786-O(strongly positve), OSRC-2(strongly positve) and SW839(weakly positve).
3. After knocking down lncRNA HOTAIR, OSRC-2 cell showes no apparent alteration of the recruitment ability towards HUVEC cell, but overexpression of lncRNA HOTAIR obviously increased recruitment of HUVEC; given the formation of tube, overexpression of lncRNA HOTAIR promotes tubeformation, while knowdown has the completely reverse effect; these phenomena are in accordance with the expression level alteration of HIF-2α and VEGFa reported.
4. SW839/shAR cell model has significantly lower ability to recruit HUVEC compared with SW839/shNC, but when it comes to tube formation, there is no obvious discrepancy between these two cell models; after knocking down AR in SW839, the decreased transcription level of HIF-2α is detected, but VEGFa; at the same time, inhibition of AR can not change the protein level of both HIF-2α and VEGFa; however, inhibiton of lncRNA HOTAIR in SW839 can decrease expression level of AR、HIF-2αand VEGFa; furthermore, knocking down lncRNA HOTAIR in weakly AR-positive cell line OSRC-2 leads to suppression of AR but this can not be detected after overexpression of lncRNA HOTAIR.
Conclusion:
1. lncRNA HOTAIR is increased in renal cell carcinoma compared to normal tissue.
2. lncRNA HOTAIR promotes vascularization via increasing the expression of HIF-2α and VEGFa.
3. lncRNA HOTAIR and AR can modulate each other, and AR has the ability to modulate vascularization of RCC, but the in-deapth molecular mechnisms needs further schoolarships.
Translated Keyword
[LncRNA HOTAIR, Renal Cell Carcinoma, Vascularization]
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