| Hepatocellular carcinoma (HCC) is one of the most common digestive malignanciesworldwide. Its malignant degree is extremely high and incidence and mortality isincreasing year by year. With high incidence, HCC places the second most lethal tumor inChina. The reason is mainly attributed to the limitations of method and early diagnosis, aswell as the frequent tumour metastasis and other varieties of complex factors. Therefore,early diagnosis is the key to improve the curative effect of the disease. Recently, many studies focus on the early diagnosis of HCC on the level of molecule or gene, thecorresponding treatment of invasion and metastasis, which may be able to herald a newstarting point of the diagnosis and treatment of HCC.With the further study on molecular mechanism of HCC occurrence and development,some RNA like microRNAs or miRNAs have attracted the attention of medical scientificresearchers. miRNAs is an abundant class of endogenous, highly conserved, smallnon-coding RNAs of18–25nucleotides in length that modulates gene expressionpost-transcriptionally. Emerging evidence demonstrates that miRNAs is closely relatedwith tumour, and mir-34a has been reported to be related to the initiation and progressionof several cancers.Notch signaling pathway exists widely in various tissue cells and is a veryconservative signal transduction in evolution. It can regulate development, apoptosis,differentiation, proliferation, and other biological behaviours. Numerous studies in recentyears show the abnormal expression of notch1exists in various tumours and is closelyrelated to the occurrence and development process of tumour. Studies have found thatnotch signaling pathway also played an important role in the occurrence and developmentof HCC.It has been found that in some tumors, mir-34a can inhibit Notch1and play thefunction of tumor-suppressor; however, the relationship between mir-34a and notch1inHCC has not been reported yet.In this study, we observe the expression and interaction of mir-34a and notch1inhepatocellular carcinoma tissues, paraneoplastic normal tissues, normal liver tissues, HCCcell lines and non tumor liver cells and explore the relationship between the mir-34a andnotch1in hepatocellular carcinoma. This study will provide basis for the research on HCCinvasion and metastasis as well as for clinical diagnosis, treatment and prognosisjudgment of HCC.ObjectiveThis study is supposed to observe the expression difference of mir-34a and notch1and furthermore their changes after regulation in hepatocellular carcinoma, paraneoplasticnormal tissues, normal liver tissues, HCC cell lines with diversified invasion abilities aswell as non tumor liver cells; research and explore their relationship with theclinicopathological features of HCC and functions in HCC invasion and metastasis.MethodsReal-time quantitative PCR (qRT-PCR) and Immunohistochemical staining (SPmethod) were used to detect the expression of mir-34a in132cases of hepatocellularcarcinoma and paraneoplastic tissues, as well as49cases of normal liver tissue;statistical methods was applied to study the relationship between mir-34a, notch1receptorexpression and clinicopathological features of hepatocellular carcinoma; Patients weregrouped to carry through Kaplan-Meier analysis so as to compare the survival time ofeach group; clinicopathological parameters were involved in Stepwise COX proportionalhazards regression model analysis; Observe the relationship between mir-34a, notch1andrelated clinicopathological features with the patients’ survival time.qRT-PCR method was used to detect the expression of mir-34a in liver tumor celllines (HepG2, Huh-7, SMCC-7721, and MHCC97) of different invasive abilities andnon-tumor liver cell line (HL-7702). Western Bloting method was used to detect theexpression of notch1in cell line.Through specific microRNA mimics and microRNA inhibitors, selected liver cancercell lines MHCC97-H, HepG2and HL-7702were acted by using transiently transfectedmethod to up-regulate or inhibit the expression of mir-34a; detect the changes of notch1expression in both of the two liver tumor cell lines by Western Bloting method.Through specific microRNA mimics, transfect liver tumor cell lines MHCC97-Htransiently to up-regulate the expression of mir-34a; transfect liver tumor cell lines HepG2transiently to inhibit the expression of mir-34a; transwell chambers matrix invasion andmetastasis test was employed to observe the changes of invasion and metastasiscapabilities of both liver tumor cell lines above. ResultsThrough qRT-PCR, the expression level of mir-34a in132cases of hepatocellularcarcinoma were significantly lower than that of the paraneoplastic tissues and normal livertissue, and metastatic carcinoma were much lower than HCC tissues, the difference wasstatistically significant (P <0.05).qRT-PCR detection showed the expression level of notch1in132cases ofhepatocellular carcinoma were significantly higher than that of the paraneoplastic tissuesand normal liver tissue, and metastatic carcinoma were much higher than HCC tissues, thedifference was statistically significant (P <0.05). Immunohistochemical staining showednotch1receptor positive staining mainly located in the cell membrane, taken on brownand brown yellow granular structure, also exists with small amount in cytoplasm. Inhepatocellular carcinoma tissues,26cases (19.70%) were negative expression,34cases(25.76%) were weakly positive,37cases (28.02%) were moderately positive, while35cases (26.52%) were strongly positive. The expression of notch1receptor inhepatocellular carcinoma (HCC) was significantly higher than that of the paraneoplastictissues and normal liver tissue, the difference of expression level was statisticallysignificant (P <0.05). In paraneoplastic tissues and normal liver tissue, few notch1receptor presented weakly positive staining, and the rest were negative. Patients weredivided into two groups based on north1score5, and we had found that mir-34a andnorth1in hepatocellular carcinoma and paraneoplastic tissues had a evident correlation,with correlation coefficients of-0.259(P=0.003) and-0.274(P=0.002) respectively.The expression of mir-34a and notch1in hepatocellular carcinoma tissues wereassociated with the malignant degree, tumors amount, tumor size, lymph node metastasisand TNM staging (P <0.05) of the patients; while not related with patient’s age, gender,liver function, tumor location, merger cirrhosis, hepatitis virus infection and AFP content(P>0.05).In accordance with the original purpose and requirements of the test, select patients with low mir-34a expression and high notch1expression, and patients with high mir-34aexpression and low notch1expression as research objects to perform Kaplan-Meieranalysis. The result showed that the three-year survival rate of patients with low mir-34aexpression and high notch1expression (11.3%) were significantly lower than of thepatients with high mir-34a expression and low notch1expression (34.7%), the differencewas statistically significant (χ2=38.163, P=0.011). The results of COX proportionalhazards stepwise regression model showed that: the larger tumor volume, multiple tumors,lower tumor differentiation, distant metastasis, venous invasion, portal vein thrombosis,tumor satellite focal, higher TNM stage, lower mir-34a expression and higher notch1expression was an independent risk factor of survival time; while otherclinicopathological factors and patient survival in this experiment acquisition time was nosignificant correlation.The detection on liver tumor cell lines with different invasive abilities by usingqRT-PCR indicated: mir-34a expression in four kinds of liver tumor cell lines (HepG2,Huh-7, SMCC-7721and MHCC97) was significantly lower than that of non-tumor livercell lines (HL-7702), among which MHCC97was of the lowest expression, while HepG2was of the highest expression. Based on the purpose and requirements of the test, the twokinds of liver cancer cell lines above were selected for further study. Detected by usingWestern Bloting method, these two kinds of liver cancer cell lines showed higher notch1expression than non-tumor liver cell line (HL-7702).Transiently transfected selected liver tumor cell lines MHCC97-H with specificmicroRNA mimics to up-regulate mir-34a expression, Western Bloting method was usedto detect the notch1expression, which was significantly weakened than before; transientlytransfected selected liver tumor cell lines HepG2with specific microRNA inhibitors toinhibit mir-34a expression, Western Bloting method was used to detect the notch1expression, which was significantly increased than before.Through specific microRNA mimics, transfected liver tumor cell lines MHCC97-H transiently to up-regulate the expression of mir-34a; results of transwell chambersinvasion and migration test showed that: the number of cells which penetrated cellmembranes was significantly reduced. Transfected liver tumor cell lines HepG2transiently to inhibit the expression of mir-34a by microRNA inhibitors, results oftranswell chambers invasion and migration test showed that: the number of cells whichpenetrated cell membranes was significantly increased.ConclusionCompared with normal liver tissues (or cell), mir-34a in hepatocellular carcinomatissues (or cells) is significantly reduced, while the expression of notch1is obviouslyincreased; mir-34a would influence the malignant biological behaviour of hepatocellularcarcinoma by retroregulating target protein notchl; the invasion and metastasis capabilitiesof liver cancer cell lines MHCC97-H and HepG2would be changed by applying specificsiRNA to up-regulate or inhibit the mir-34a expression; combined detection on expressionand changes of mir-34a and notch1in hepatocellular carcinoma would play a meaningfulpart in the clinical diagnosis, treatment and prognosis diagnosis of HCC. |
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