| Objective To investigate the expression of ribosomal protein L34(RPL34)in human squamous cell carcinoma and the effect of knocking down RPL34 gene on proliferation and apoptosis of human skin squamous cell carcinoma SCL-1 cell line.MethodsWe collected 14 cases of skin squamous cell carcinoma and 16 cases of normal skin tissues of paraffin specimens from January 2016 to January 2017 in Department of Dermatology and Sexology,Affiliated Hospital of Inner Mongolia Medical University.We taken wax blocks of squamous cell carcinoma and normal tissues,and then routinely sliced,dewaxed,sealed and antigen repaired,and then added rabbit anti-human RPL34 antibody(diluted 1:100),overnight at 4℃,adding secondary antibody for incubation for 60 min,adding fast red dye solution for dyeing for 15min,adding DAB enzyme substrate developer for color development,washing with distilled water,dyeing with hematoxylin,dehydrating and sealing.The negative control was incubated with PBS instead of primary antibody.All sections were independently read by two skin pathologists without clinical diagnosis,and the RPL34 staining intensity and positive rate of the sections were evaluated.The staining intensity grades were negative,gradeⅠ,gradeⅡ and gradeⅢ,corresponding to 0,1,2and 3 points.The positive staining rates were negative,1% ～ 25%,26% ～ 50%,51% ～75% and 76%～100%,respectively,with scores of 0,1,2,3 and 4.The expression level of RPL34=staining intensity score×staining positive rate score.The expression of RPL34 gene in cutaneous Squamous Cell Carcinoma was analyzed by immunohistochemistry in 14 c SCC patients and 16 normal controls.Next,sh RNA-lentivirus was used to knockdown RPL34 expression in the c SCC cells SCL-1.the knockdown efficiency of target gene was detected by q PCR,and the expression level of RPL 34 was analyzed by Western blotting.Celigo cell count was used to detect cell growth.PI-FACS was used to measure cell cycle distribution and apoptosis,and MTT assay was also performed(All experiments were repeated 3 times,and the average value was taken).Chi-square test was used to compare the counting data,and the mean±standard deviation was used to express the measurement data,t-test or rank sum test was used to compare the differences between the two groups.When α was set to 0.05,P<0.05 was considered to be statistically different.ResultsImmunohistochemical study showed that the cytoplasmic expression score of RPL34 was significantly higher in the c SCC tissues(2.143 ±1.956)than in the normal control tissues(0.500 ±0.516,z=3.53,P<0.05).RT-PCR showed that the relative mRNA expression of RPL34 in the SCL-1 cells was significantly lower in the sh RNA group(0.149±0.016)than in the control group(1±0.018,t=36.95,P<0.05);Western blotting analysis revealed that the relative protein expression of RPL34 in the SCL-1 cells was significantly lower in the sh RNA group than in the control group.Compared with the control group,the sh RNA group showed significantly increased proportion of S-phase cells(t=13.76,P<0.05),but significantly decreased proportion of G1-phase cells(t=36.62,P<0.05)and no significant change was found in G2/M phase(P>0.05).The apoptosis rate was significantly higher in the sh RNA group(9.42% ±0.16%)than in the control group(4.58%±0.41%,t=19.02,P<0.05).MTT assay showed that the cell viability was significantly decreased in the sh RNA group(0.815±0.005)than in the control group(1.886±0.005,t=265.91,P<0.05)at 120 hours in the experiment.Conclusions1.through immunohistochemical screening,the expression of RPL 34 in c SCC tumor tissue was significantly higher than that in normal skin tissue,it has statistical significance.2.RNA interference test mediated by lentivirus infection,after knocking down RPL34,inhibition of proliferation and increase of apoptosis of human squamous cell carcinoma SCL-1 cell,the number of cells in S phase increased,and the number of cells in G1 phase decreased,these findings further confirm that RPL34 plays an important role in tumor cell proliferation,differentiation and apoptosis,RPL34 may be a potential therapeutic target for skin squamous cell carcinoma. |
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