| AimOn the basis of the importance of miRNA in cancer development, the study is under the guidance of combination ofexterior-interiormeridianacupoints selection to intervene HepG2 nudemouse transplanted cancer model by electro-acupuncturing "ST36+SP 6". Use the research main line of miR-409-5p targeted inhibition TGF-?1, we explore the regulation mechanism of miR-409-5p targeted inhibition TGF-?1 for electro-acupuncture felicitous repression of cancer effect in both cell and in-vivo experiment levels. The study reveals the action principle of felicitous repression of cancer through electro-acupuncture more comprehensively and provides a new scientific evidence to clinical apply ofacupuncture in cancer diseases.MethodsThis study is analyzed through 3 levels:effect validation of cancer control via electro-acupuncture, verification of target gene TGF-?1 filtered and predicated by miR-409-5pexpression profile and miRNA-409-5ptargeted inhibition TGF-?1 molecular regulation mechanism of felicitous repression of cancer effect by electro-acupuncture. The analysis will be implemented in 5 steps.1.32 nude mice are divided into electro-acupuncture group, model group, electrical stimulation group and blank control group randomly,8 mice per group. Establish HepG2 nude mouse subcutaneous transplanted cancer model and intervene "ST36+SP 6"in both lower extremities of nude mouse via electro-acupuncture or direct electrical stimulation (30 mins/day,10 days/a treatment course) to observe felicitous repression of cancer effect through electro-acupuncture.2. Run gene expression spectrum detection of nude mouse transplanted cancer tissue under electro-acupuncture intervention through LC Sciences miRNAs chip to filter miRN As differential expression. Furthermore use qRT-PCR on miR-409-5p which has further research value to expand the results of sample validation chip.3. The target gene with significant clinical pathological features is obtained by the related algorithm calculation of LC Sciences Company through database search in Target Scan, Pic Tar and miRanda. Refer to GO and KEGG analysis to establish the network connect table of miRNAs-KEGG-network with most research value. Then predict the target gene which possesses further research value in our research through literature consulting in database access.4. Inspect the HepG2 cell proliferation and migration influence by miR-409-5pmimics via MTT method and scratch test and predict the expression quantity of target gene TGF-?1 mRNA through qRT-PCR detection.5. Employ the antagomir-409-5pinjection technology to seal the effect of miR-409-5p. 32 nude mice are randomly divided into electro-acupucture+antagomir-409-5p group, electro-acupucture+antagomirNcontrol group, model group and blank control group. Check the nude mouse serum TGF-?1 content, immunohistochemicaldetetion of translated cancer tissue TGF-?1 and PCN A expression.Results1. The cancer weight ratio in electro-acupuncture group is superior to it in model group and electrial stimulation group and a statisticallysignificantdifferences (P<0.05). There is significantdifferences between groups in cancer inhibitory rate (P<0.05), electro-acupuncture group is better than electrial stimulation group. Electro-acupuncture has better inhibition effect to the cancer growth rate than the other groups.2. The screening results of miRNAs differential expression with the upper limit of P value<0.05 are gained by analyzing 14 miRNAs genes in differential expression including 10 differential expression genes due to the miRNAs of transplanted cancer sample escalation, among which the p value of miR-409-5p?0.01. qRT-PCR detection method verifies the expansion rate of miR-409-5p in electro-acupuncture group is 1.00 while it in model group is 0.24. The screening result in miR-409-5p expression of electro-acupuncture group is matched with chip filtering result.3. It is predicted by 3 databases such as Target Scan that TGF-?1 is the target gene of miR-409-5p. The analysis of GO and KEGG also proves the vital effect of TGF-?1 in miR-409-5p related cancer pathways.Because TGF-?1 is the important cytokines in the developmentof cancerand cancer immunosuppression microenvironment, so is closely related to inhibition effect to the cancer growth ofelectro-acupuncture.4. MTT shows that miR-409-5p can restrain cellproliferation of HepG2 cell expression and its regulative ability is proportional to the concentration of miR-409-5pmimics and time increase. Scratch it proves that the transwell migration ability of miR-409-5p mimics is much lower than NC mimics group and miR-409-5p in HepG2 cell expression can debase the transwell migration ability. The qRT-PCR detection method verifies that miR-409-5pmimics can obviously suppress the expression level of TGF-?1 mRNA.5. There are significant differences in all felicitous repression of cancer effect indicators such as cancer weight ratio, and cancer inhibitory rate serum TGF-?1 content, transplanted cancer tissue TGF-?1 and PCNA expression level between groups (P<0.05), the model group is the worst in the 3 groups. Then the cancer weight ratio, cancer weight ratio and transplanted cancer tissue TGF-?1 expression level in Ncontrol group is superior to it in antagomir-409-5p group (P<0.05).Conclusions1. Electro-acupuncture has a inhibition effect to the cancer growth rate of nude mice.2. miR-409-5p can participate in molecular regulation of cell proliferation and influences the migration ability of cancer cell.3. Electro-acupuncture felicitous repression of cancer effect has a possible and compact relevance with miR-409-5p targeted inhibition to TGF-?1 molecular regulation mechanism. |
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