| Objective To investigate the effects of the recombinant lentiviral vectorfor RNAi（RNA interference）of MCM7gene on the expression of MCM7geneand on the proliferation and apoptosis of human liver cancer SMMC-7721cellsand the growth of subcutaneous tumor of human liver cancer SMMC-7721innude mice and its possible mechanisms.Methods1.The expression of MCM7mRNA were detected by reversetranscription and quantitative real-time polymerase chain reaction (qRT-PCR) inSMMC-7721and HepG2, SK-hep-1of these three kinds of human liver cancercells to screen experiments using cells.2. Four vectors carrying shRNA targeting the MCM7gene (MCM7-shRNAexpression vector) were constructed by RNA interference technology, and thentransfect stably to human liver cancer cell lines SMMC-7721, and then screenedeffective targets. 3. Human liver cancer SMMC-7721cells were seeded in6well plates anddivided into three groups: the experimental group, the normal control group andthe negative control group. For the experimental group, Liver cancerSMMC-7721cells were transfected with the recombinant lentivirirus vector(LV-shRNA-MCM7), while the negative control with an control lentiviral vector(LV-shRNA-NC), the normal control with no treatment. The mRNA and proteinlevel of MCM7were analyzed by transcription polymerase chain reaction(RT-PCR), quantitative real-time polymerase chain reaction (qPCR), andWestern blot, and then determined the interference efficiency to got the effectivetarget.4. For the cells of three groups, cell proliferation was detected by MTTassay; cell colony formation was detected by Giemsa staining. The cell cycleand apoptosis were observed by flow cytometry (FCM).5. All the transfected cells were then selected through puromycin.30BALB/c nude mice were randomly divided into three groups，10mice in eachgroup．Nude mice were inoculated subcutaneously with cells of each group toestablish the subcutaneous tumor model of hepatocellular carcinoma. Observethe nude mice into tumor, transplanted tumor growth and tumor growth curvedrawing. Tumor volume and weight were estimated after four weeks.Theexpression of MCM7gene were analyzed by reverse transcription andquantitative real-time polymerase chain reaction (qRT-PCR), Western blot andimmune histochemical method.Result1. The reverse transcription and quantitative real-timepolymerase chain reaction (qRT-PCR) results showed that the expression ofMCM7mRNA in human liver cancer cells SMMC-7721were the highest inthree cells. And the gene abundance of cells expressed were suitable for knocking subtraction verification. And then choose SMMC-7721cells as theexperimental group.2. Successful building MCM7-shRNA expression vectors, and has beenverified by sequencing and correct. After transfecting SMMC-7721cells, cellfluorescent display rate>90%, endogenous targets were to be confirmed.3. MCM7mRNA and protein expression of four MCM7–shRNA targetsgroups were all above50%than the negative control group and the normalcontrol group, among them with LV-shRNA-MCM7(4) interference target onthe highest efficiency, reduction of88.95%,87.89%and82.25%,88.95%respectively (P<0.05), as the experimental group.4. Determined by MTT method, the results showed that the experimentalgroup of cells at490nm absorption luminosity values in24,48,72and96hourafter transfected was lower than the negative control group and the normalcontrol group, the difference is significant (P<0.05). At the same time, Giemsastaining results showed that the clone formation rate of LV-shRNA-MCM7group (6.00%±0.50%), significantly lower than the blank control group(14.10%±0.36%), and the negative control group (13.73%±0.17%),significantly suppressed experimental cell growth (P<0.05). Flow cytometryanalysis showed that the percentage of cells in G1phase increased in cellstransfected with the MCM7-shRNA (P<0.05). The apoptosis rate of theexperimental group was (22.27%±1.22%), significantly higher than the negativecontrol group (0.05%±0.07%), and blank control group (0.03%±0.06%).Compared with the negative control group and the normal control group, there isan obvious apoptosis of the experimental group (P<0.05).5. Cancer cells in nude mice inoculated with the first four days after in theblank control group and negative control group had tumor formation, and delayed two days after the experimental group had tumors in nude miceformed.To6days after inoculation of cells all had tumor formed. Comparedwith the normal control group and the negative control group, the tumor growthof experimental group slowed significantly（P＜0.05）.The mean volume ofexperimental group, negative control group and normal control groupwas(27.72±7.80)mm3,(81.86±10.91)mm3and (79.75±16.61）mm3; the meanweight was(0.19±0.06)g,(0.501±0.14)g and (0.509±0.18)g, respectively. Theexperimental group was different from negative control group and normalcontrol group in the mean volume and the mean weight (P＜0.05). And theMCM7mRNA and protein expression of transplanted tumor tissues in theexperimental group were significantly decreased (P＜0.05).Conclusion1. The recombinant lentiviral vector for RNAi （RNAinterference）of MCM7gene can effectively suppress the mRNA and proteinexpression of MCM7gene.2. RNAi-induced MCM7down-regulation could better inhibit the cellgrowth of human liver caner SMMC-7721cells, suppress the formation of cellcloning, block the cell cycle at G1phase, and induce cell apoptosis.3. RNAi-induced MCM7down-regulation could better inhibit the growth oftransplanted tumor.4. It suggests that MCM7may be a new useful target for genetherapy ofhepatocellular cancer in future. |
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