Research On Differentially Expressed Long Noncoding RNAs In Hepatocellular Carcinoma Cell Lines With Different Lung And Lympha Node Metastasis Potential

Background and aims:Hepatocellular carcinoma （HCC） is one of the most common malignancies and the second cause of cancer-related deaths worldwide. Although it has made great progress in diagnosis and treatment of HCC, the efficacy of treatment for HCC is still far from satisfactory. Metastasis is the main obstacle of ameliorating the poor prognosis of HCC. HCC cells spreads via the hematogenous route, the lymphatic route, or by direct invasion into adjacent organs. HCC cells develop the ability to colonize the specific organ, while HCC cells are prone to metastasize more frequently to lung than to lympha node. It has been well recognized that specific metastatic seeding and growth is determined by the intrinsic genetic properties of cancer cells.By definition, long non-coding RNA （lncRNA） are transcripts>200nucleotides [nt] in length. LncRNAs were first considered to be "transcriptional noise" without any biological function. While, the recent reports indicated lncRNAs could regulate the mode of transcription and the activity of protein, be as precursor of microRNA, change the process of RNA and maintain the structures and order of cells. Meanwhile, more and more evidence indicated that IncRNAs disregulated in many diseases, especially in cancer, which attracted great interests of researchers. However, there are still little reports about the function of IncRNAs in metastasis of liver cancer. In our study, expression profiles of two HCC cell lines （HCCLYM-H2and HCCLM3） were analysed using8660K Arraystar Human LncRNA Array v2.0to understand better the function of lncRNAs in organ-specific metastasis of HCC.Methods:In current study, we used the method of in vitro monoclonal culture and in vivo consecutive selection to optimize the HCC cell line HCCLYM-H, and finally get cell lines named HCCLYM-H2, which showed stable lymph node metastatic potential. Meanwhile, established the lympha node metastasis model in nude mouse with this cell line. Arraystar Human LncRNA Array v2.0containing33,045IncRNAs and30,215mRNAs was used to obtain expression profiles from two HCC cell lines HCCLYM-H2（high lympha node metastatic potential） and HCCLM3（high lung metastatic potential）. Agilent Feature Extraction software （version11.0.1.1） was used to analyze the data. To identify lncRNAs with potential functions in metastasis of HCC, we performed CNC network. Six lncRNAs （BC070168, AK054728, uc001szi.3, ENST00000489452, ENST00000436499, uc004cff.2） and two mRNAs （NM₀00728and NM₀04616） were further validated using qRT-PCR.Results:1. We obtained25monoclonal strains as number1-25. Nine of these were well-grown for further2rounds of selection, and finally one monoclonal strains with highest lympha node metastasis and lowest lung metastasis were selected as HCCLYM-H2. The results of pathological examination showed the rate of lympha node and lung metastasis were100%and20%, respectively.2. The tumor formation rate of HCCLYM-H2cells seeded into mice foot pad was100%, and after5weeks the maximum diameter of primary tumors was1.5cm. After5weeks of liver orthotopic transplantation, we could observe the distant metastases of para-aortic lymph node, common iliac lymph nodes, intercostal lymph node, and so on.3. The expression profiles in paired samples were shown by calculating log fold change HCCLYM-H/HCCLM3（H/LM3）. A total of1629mRNAs and1483lncRNAs were differentially expressed between HCCLYM-H2and HCCLM3significantly （p≤0.05,≥1.5-fold change）. The results showed306lncRNAs and717mRNAs were up-regulated in HCCLYM-H2cell lines compared with HCCLM3cell Iines, and1177lncRNAs and912mRNAs down-regulated. LncRNA RP11-672F9.1（ENST00000450980） was the most up-regulated lncRNA （log2fold change9.543524）. LincRNA-TSPAN8（BC070168）（log2fold change65.74432） and lincRNA-CALCA （AK054728）（log2fold change57.2131） were the most down-regulated lncRNAs, which were associated with the protein coding gene TSPAN8and CALCB, respectively. Interestingly, the fold change of mRNAs TSPAN8and CALCB was equally high （48.5841and74.1006, respectively） according to the data of microarray.4. The structure of the CNC networks of lung metastatic and lympha node metastatic cell lines were significantly different, indicating that the inter-regulation of lncRNAs and mRNAs varied in different metastatic potential cell lines. In the HCCLYM-H2CNC network,132IncRNAs and306mRNAs were linked by1462 edges. Nearly680edges （1462;46.51%） connected mRNAs, and647edges （44.25%） connected mRNAs and lncRNAs, whereas another135edges （9.23%） linked pairs of lncRNAs. Meanwhile,173lncRNAs and398mRNAs were linked by3115edges in HCCLM3CNC network. About1481edges （3115;47.54%） connected mRNAs,1293edges （41.51%） connected mRNAs and lncRNAs, and another341edges （10.95%） linked pairs of lncRNAs.5. The results of real time qPCR showed the fold change of ENST00000436499in HCCLYM-H2cell line compare with HCCLM3cell line was3.7885±0.3855, and BC070168, AK054728, uc001szi.3, ENST00000489452, uc004cff.2in HCCLM3cell line compared with HCCLYM-H2cell line were69.7498±6.3728.15.6455±2.5898.13.0636±3.7768.7.0798±3.6812.7.1488±2.3995, which were consistent with microarray data.Conclusions:1. Establish a cell line with high lympatic metastatic potential and specific metastasis model in nude mouse.2. This is to our knowledge the first time that the expression profiles of lncRNAs have been studied with regard to organ specific metastasis in HCC, and we identified a large number of lncRNAs that related to organ specific metastasis of HCC.3. We found multiple lncRNAs related to organ-specific metastasis of HCC, CNC network indicated these lncRNAs were also associate with the occurrence and progression of tumor as well as cell adhesion and cell migration.