| ?Background? Hepatocellular carcinoma is one of the most commonly malignant tumor.According to the the epidemiological investigation,the number of hepatocellular carcinoma patients newly diagnosed has reached 780000 people in 2012,and the figure is still rising today.Because its early diagnosis is difficult,diagnosis is almost late stage.When it is early stage,invasion and metastasis happened which induced bad prognosis.At present,the treatment mainly depends on surgery,interventional therapy,chemotherapy and Sorafenib that represented small molecular targeted drugs.Due to the complicated mechanism of occurrence and less sensitivity to most chemotherapy and target drugs,it is quite difficult for HCC patients to benefit long survival time.There is still a long way to go for exploring the HCC treatment.Therefore,the in-depth study of molecular mechanism of liver disease will be benefit to grasp the key molecular in the process of liver cancer development,develop new anti-cancer drugs,and it will be of great significance to improve the prognosis of patients.Lnc RNAs has been discovered to widely existed in genome in recent years.Unlike coding protein gene,lnc RNAs lack specific open reading frame that encode proteins.Because of this,it had been regarded as "noise" of genome sequence or "dark matter" for long time.With the deepening of the research,a number of studies have confirmed that lnc RNAs has abnormal expression in the vast majority of human tumors.It is widely involved in the occurrence and development of tumor and the drug resistance and other important process.Lnc RNAs regulate gene transcription and and translation through a variety of mechanisms,and has effect on the cell biology behavior of cancer.Lnc RNAs also positively or negatively regulate the cell biology behavior of HCC.Because of the numerous types and complicated regulatory mechanism,there is still a little study of existing research on interaction between HCC and lnc RNAs.Therefore,to explore the function and molecular mechanism of HCC related lnc RNAs will contribute to the overall understanding of complex biological behaviour regulation,provide new screening and treatment targets for HCC.?Aims? To screen the lnc RNAs closely related to other tumor,we try to find lnc RNAs which are differentially expression in both HCC tissue and cell line.We need do research its correlation with the clinical pathological characteristics and prognosis.Through establishing the cell model of lnc RNAs downregulation,we carry out research the function of lnc RNAs in regulating hepatocellular carcinoma cell biology behavior.To further explore the specific molecular regulation mechanism of candidate lnc RNAs molecules on liver cancer cell biology behavior,so as to improve the liver cancer cell biology behavior regulation network signal,to provide screening and new therapeutic targets of HCC,as well as the new experimental foundation and theoretical foundation for Lnc RNAs.?Methods? 1.We brought 21 lnc RNAs closely related to other kinds of tumor into our research through reviewing literature,and detected expression of lnc RNAs in three kinds of HCC cell lines and normal immortalized HCC cell line by q RT-PCR.To secreen the candidate lnc RNAs in 47 HCC tissue and adjacent tumor tissue by q RT-PCR.All patients were divided into high expression and low expression group based on the expression of candidate lnc RNAs in HCC tissue.We analyzed whether there was difference about clinical pathological characteristics and prognosis between groups.2.We chose Hep G2 cell line as the object of our research.We established the cell line in that lnc RNAs had stable downregulation.MTT was used to compare the growth curve difference among negative control group,blank control group and interference group.Plate clone formation assay was used to detect the rate of clone formation among three group.Ed U was used to detect the DNA replication activity.Nude mouse transplantation tumor experiment was used to detect the change of volume and weight of tumor.All of these experiment aimed to evaluate the influence of lnc RNAs on proliferation.Wound healing assay was used to detect the invasion ablity of cells.Annexin V-FITC was used to detect the apoptosis of cells,and westernblot was used to detect the expression of bax and bcl2 which are apoptosis related protein.3.We analyzed the possible target gene of lnc RNAs through the gene location and analysis of biological imformation.Whether target gene had a differential expression on m RNA and protein level in tissue and cell were detected through q RT-PCR,westernblot and immunohistochemistry,and were analyzed the relevance with the expression of lnc RNAs.Through tranfecting the overexpression vector into lnc RNA downregulated cell lines,we used MTT,Ed U,Annexin V-FITC and westernblot to evaluated whether the lnc RNA played a role in the proliferation and apoptosis through target gene 4.Abnormal signaling pathway involved in lnc RNA and target gene was inferred based on biological imformation.The expression of key protein of this signaling pathway was dected in lnc RNA downregulated cell lines and target gene overexpressed cell lines.Whether lnc RNA and target gene regulated the HCC cell biological behavior through this signaling pathway were evaluated by using agonist and specific inhibitor of pathway.?Results? 1.Through analyzing 21 kinds of lnc RNAs the differential expression between HCC cell lines and normal immortalized liver cells,we found that nc RAN-L and UCA1 might be candidate lnc RNA.We focused on nc RAN-L,and there was a same differential expression between HCC tissue and adjacent tumor tissue.Moreover,high expression of nc RAN-L group had much more huge tumor volume(?2=8.563,P=0.003)and more number of tumor(?2=6.940,P=0.008)compared with low expression group.It seemd had no relation with gender,age,the level of AFP,lymph node metastasis and distal metastasis.The result of Kaplan Meier survival analysis suggested that patient of low expression of nc RAN-L had longer survival time compared with ones of another group(Log-rank ?2= 4.439,P=0.351).The result of Cox mulitivariables analysis suggested that the expression level of nc RAN-L could be the independent prognosis factor of hepatocellular carcinoma patients.The HR of nc RAN-L was 2.347 of which confidence interval was 1.091-5.048,and P-value was 0.029.2.Through detecting the transfection efficiency of the small interfering RNA,we choose si-RA1 as interference fragment of subsequent experiments.We succeed in establishing the nc RAN-L stably downregulated Hep G2 cell line also known as Hep G2/si-RA1 with lentivirus.The result of MTT suggested nc RAN-L downregulated could inhibit the proliferation.The result of plate clone formation suggested nc RAN-L downregulated could suppress the ability of clone formation.The result of Ed U suggested nc RAN-L downregulated could suppress the DNA replication activity.Nude mouse transplantation tumor experiment suggested nc RAN-L downregulated could shrink the volume and weight of tumor.However,Wound healing assay had no positive result.The result of Annexin V-FITC suggested si-RA1 group has higher apoptosis rate of HCC cell compared with two control group(vs.si-NC,7.72%±1.15;vs.control 5.42%±0.68).The expressions of bax and bcl2 also supported this viewpoint.3.We found that ST6GALNAC2 located in the 4.3kb distance from the downstream of nc RAN-L,which had relationship with the cell biological behavior of breast cancer and thyroid cancer.Therefore,we inferred it might be the target gene of nc RAN-L.The levels of m RNA and protein of ST6GALNAC2 had a significant difference in tissue and cell lines.The expression of ST6GALNAC2 had a posistive correlation with nc RAN-L in tissue(r=0.3674,P=0.0111).There was a lower expression of ST6GALNAC2 in Hep G2/si-RA1 compared with control group.It suggested that nc RAN-L might control the expression of ST6GALNAC2.The result of MTT,Ed U,Annexin V-FITC,and Westernblot suggested overexpression of ST6GALNAC2 in Hep G2/si-RA1 could reverse the proliferation reduced by knock-out nc RAN-L and strengthen the DNA replication activity and restore the resistance to apoptosis to some extent.4.We found that the downregulation of expression of nc RAN-L could significantly suppress the expression of p-akt.However,overexpression of ST6GALNAC2 on this basis could enhance the expression level of p-akt.It preliminarily suggested that nc RANL/ST6GALNAC2 might be involved in the abnormal PI3K/Akt pathway of HCC cell.To further clarify the function of nc RAN-L,we pour PI3K/Akt agonist Igf-1 and specific inhibitor LY294002 into Hep G2/si-RA1 and ST6GALNAC2 overexpressed Hep G2/siRA1,respectively.The result showed that Igf-1 could restore the ability of proliferation and resistance to apoptosis reduced by knock-out nc RAN-L.Moreover,LY294002 could inhibit the proliferation and counter the resistance to apoptosis enhanced by ST6GALNAC2 overexpressed.All of these results further explicited nc RANL/ST6GALNAC2 involved in PI3K/Akt of HCC cells.It is also one of possible mechanism of nc RAN-L regulating proliferation and apoptosis of HCC cells.?Conclusion? 1.We found lnc RNA nc RAN-L had higher expression in HCC tissue and cell lines.The expression of nc RAN-L had correlation with clinical pathological characteristics and prognosis.It suggersted that nc RAN-L could play a role of oncogene in the tumorigenesis and development of HCC.Nc RAN-L could be potientially independent factor for assessment of clinical outcomes in HCC patients.2.Nc RAN-L could regulate the HCC cell biological behavior through upregulating proliferation and inhibiting apoptosis via bcl2/bax pathway.Nevertheless,it had no business with invasion of HCC cell.3.ST6GALNAC2 was one of target gene of nc RAN-L.Nc RAN-L activated the PI3K/Akt signaling pathway via upregulating the expression of ST6GALNAC2,which regulated the proliferation and apoptosis of HCC cells.It illuminated the novel mechanism between lnc RNAs and cell biological behavior of HCC.It also provided theoretical basis and experimental basis for exploring new clinical therapy target. |
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