长非编码RNA（long-noncoding RNA，lncRNA）类型众多，且作用方式复杂多样，可以与DNA、RNA和蛋白等结合发挥 “支架”、“引导” 、“海绵”等等作用，对基因表达的调控具有很高的精密性。精子发生 （Spermatogenesis）是极其复杂 而特异的细胞分化过程，相较于体细胞的分化，历经了许多重要的特殊事件，这些事件都在精密的调控下进行。因此，lncRNA在精子发生中很有可能也扮演着重要调控者的角色。但是，至今lncRNA在精子发生中的功能与机制研究还比较少。所以，深入研究lncRNA在精子发生过程中的功能与机制，对解决男性不育这世界性医学难题将具有重要意义。

有研究表示RNA结合蛋白QKI可以和相应lncRNA结合发挥功能，在神经发育、血管发育和肿瘤发生等领域，具有调控细胞的增殖、分化、迁移等作用。本实验室前期研究发现QKI5在睾丸组织中表达水平相对较高，尤其在4 w和5 w的睾丸组织中，主要在粗线期精母细胞表达水平最高；在细胞系上发现QKI5在GC1-spg中表达水平较高， 主要定位于细胞核，部分位于细胞质中。RIP-seq和生信分析发现，在MAPK信号通路上高富集，而此信号通路大多与细胞凋亡、增殖和分化相关。

本研究在此基础上，着重开展QKI5在精子发生中的功能。我们首先探索了QKI5在细胞凋亡中的作用。利用siRNA抑制GC1-spg细胞中内源QKI5蛋白的表达，分别在0 h、3 h、6 h、9 h、12 h加入Etoposide诱导细胞凋亡，采用流式细胞仪检测GC1-spg细胞凋亡的比例变化。实验结果发现，随着Etoposide诱导时间增长，对照组和实验组的凋亡比例都在增加，但是实验组的细胞凋亡比例明显低于对照组。表明抑制QKI5的表达水平可以抑制GC1-spg细胞的凋亡，即QKI5在生精细胞中对细胞凋亡有促进作用。实验还发现si-QKI5实验组的cleaved caspase3表达量变化明显，且作为caspase3凋亡前期的p38-MAPK信号通路中p-p38、p38等关键分子及其信号通路上游PARP蛋白的表达水平与对照组形成明显差异。表明在生精细胞中QKI5是通过活化p38-MAPK信号通路来促进细胞凋亡。

前期通过RIP、RNA-pulldown和生信分析，最终筛选出QKI5结合的13条候选lncRNA。其中lnc6和lnc11有组织特异性，与QKI5的变化趋势大体一致，并将目标最后指向lnc6。本研究中采用RNA-pulldown进行反向验证，结果显示反义链探针可以同时检出lnc6和QKI5，表明lnc6与QK5相互结合，确认了lnc6为QKI5结合的长链非编码RNA。

随即开展了对lnc6的功能认识。在不同组织、六种不同时期生精细胞、不同发育时期的睾丸组织、四种不同生殖细胞系和细胞定位等检测了lnc6的表达量，发现lnc6在睾丸组织中表达量相对较高，尤其前期生精细胞表达量最高，定位在细胞核。我们用siRNA抑制GC1-spg细胞内源lnc6的表达，再加入Etoposide处理细胞诱导细胞凋亡，检测GC1-spg细胞凋亡的比例变化。实验结果发现，随着Etoposide诱导时间增长，对照组和实验组的凋亡比例都在增加，但是实验组的细胞凋亡比例明显高于对照组，表明了抑制lnc6的表达可以促进GC1-spg细胞的凋亡；实验组的PARP蛋白、p-p38蛋白、p38蛋白的表达水平也随之发生了相应变化。结果显示在生精细胞中lnc6是可以抑制GC1-spg细胞的凋亡，是通过抑 制p38-MAPK信号 通路来实现的，这与QKI5的作用正好拮抗。

进而探索QKI5和lnc6是如何调控细胞凋亡的？我们发现当抑制lnc6时，p38-MAPK信号通路中的mapk11、mapk12和mapk14的RNA稳定性增高；而当抑制QKI5表达时，lnc6的表达量相比对照组有微弱上升趋势；当过表达外源性lnc6，发现 cleaved caspase3、p-p38、p38和PARP等蛋白的表达量低于对照组；而同时抑制lnc6和QKI5时，也发现si-lnc6/QKI5实验组的表达量介于si-lnc6、si-QKI5组之间，与NC对照组有大致相同趋势。这些实验结果也验证了以下推测：正常情况下，QKI5可以维持p38-MAPK的mRNA和lnc6的稳定性；当QKI5缺失时，p38-MAPK的mRNA不能被QKI5稳定，故而不能正常翻译，从而使得cleaved caspase3表达被抑制，细胞凋亡过程被抑制；当lnc6被抑制时，QKI5与lnc6结合位点被大量释放，QKI5与p38-MAPK的结合位点增多，从而使得p38-MAPK的mRNA被QKI5稳定，使得cleaved caspase3表达被激活，从而促进细胞凋亡。p38-MAPK的mRNA与lnc6可能通过竞争性结合QKI5的结合位点从而调控细胞凋亡。

综上，我们在体外研究了QKI5及其结合的lnc6在精子发生中的功能和机制：发现QKI5可以通过活化p38-MAPK信号通路来促进细胞凋亡；同时确认了lnc6可与QKI5结合，其可以通过抑制p38-MAPK信号通路来抑制细胞凋亡；QKI5与lnc6的作用机制可能是通过竞争调节模式来调控细胞凋亡。

在研究QKI5对细胞凋亡影响的同时，我们还利用CRISPR/CAS9技术构建了qki基因敲除的GC1-spg细胞株，通过筛选和鉴定，获得1# 靶点单细胞株3株，2# 靶点单细胞株3株，最终选择GTCCGAG的碱基缺失突变体1#06作为研究细胞增殖和分化实验的材料。结果发现cas-QKI5实验组细胞增殖明显慢于对照组的细胞增殖趋势（*P < 0.05）；检测减数分裂相关分子标志物c-kit、Mtl5和Hspa2的表达水平，发现实验组显著低于对照组。这表明QKI5也具有促进细胞增殖和细胞分化的功能。

Long non-coding RNAs (lncRNAs) have various types and functions in various ways. It can combine DNA, RNA, and protein to function as a "stent," "guide," and "sponge," and express genes. Supervision has a high degree of accuracy. Spermatogenesis is a very complex and specific cell differentiation process. Compared with the differentiation of somatic cells, it has undergone many important special events. These events are precisely controlled. Therefore, lncRNA may play an important regulatory role in spermatogenesis. However, the role and mechanism of lncRNA in spermatogenesis have not been studied so far. Therefore, an in-depth study of the roles and mechanisms of lncRNAs in the process of spermatogenesis is of great significance in solving male infertility problems. This is a worldwide medical problem.

Studies have shown that RNA-binding protein QKI can act in conjunction with the corresponding lncRNA, which can regulate cell proliferation, differentiation, and migration in areas such as neural development, vascular development, and tumorigenesis. Our previous study found that the expression level of QKI5 in the testis is relatively high, especially in the testis of 4w and 5w, mainly in the thick line sperm cells; the expression of QKI5 in the cell line is the expression level of GC1- in the spg. Higher, mainly located in the nucleus and partly in the cytoplasm. RIP-seq and bioinformatics analysis revealed high enrichment of the MAPK signaling pathway, which is mainly associated with apoptosis, proliferation, and differentiation.

Based on this research, we focused on the role of QKI5 in spermatogenesis. We first explored the function of QKI5 in apoptosis. siRNA was used to inhibit the expression of endogenous QKI5 in GC1-spg cells, and etoposide was added at 0, 3, 6, 9, 9, 12 hours to induce apoptosis. . The proportion of apoptosis in GC1-spg cells was measured by flow cytometry. The experimental results showed that with the increase of etoposide induction time, the proportion of apoptosis in the control and experimental groups increased, but the proportion of apoptosis in the experimental group was significantly lower than in the control group, indicating that inhibition of QKI5 expression can inhibit GC1-spg cells Apoptosis in turn indicates that QKI5 promotes spermatogenic cell apoptosis. The experiment also found that the expression level of cleaved caspase3 in the si-QKI5 experimental group changed significantly and was a key molecule in the p38-MAPK signaling pathway, such as the expression level of p-p38, p38, PARP protein in the upstream, and caspase3 apoptosis in the control group. group. There are significant differences. The results showed that QKI5 promoted spermatogenic cell apoptosis by activating the p38-MAPK signaling pathway.

In the previous analysis, 13 candidate lncRNAs bound by QKI5 were screened by RIP, RNA pull-down and bioinformatics analysis. Among them, lnc6 and lnc11 have tissue-specificity, which is basically consistent with the change trend of QKI5, and the ultimate goal is to lnc6. In this study, RNA pulldown was used for reverse validation. The results showed that the antisense strand probe can detect lnc6 and QKI5, indicating that lnc6 binds to QK5, confirming that lnc6 is a QKI5-bound long-chain non-coding RNA.

Immediately carry out the function understanding of lnc6. The expression of lnc6 was detected in different tissues, six testicular tissues, testicular tissues at different stages of development, and four different germline and cell-specific spermatogenic cells. The expression of lnc6 in testis is relatively high, especially early. Spermatogenic cells express the highest amount, located in the nucleus. We used siRNA to inhibit the expression of endocrine lnc6 in GC1-spg cells and then added etoposide to treat the cells to induce apoptosis and detect the proportion of apoptosis in GC1-spg cells. The experimental results showed that with the increase of etoposide induction time, the proportion of apoptosis in the control and experimental groups increased, but the proportion of apoptosis in the experimental group was significantly higher than the control group, indicating that inhibition of lnc6 expression can promote GC1-spg cells Apoptosis; PARP protein, p-p38 protein and p38 protein expression levels in the experimental group also changed accordingly. The results showed that lnc6 inhibited the apoptosis of GC1-spg cells in spermatogenic cells by inhibiting p38-MAPK signaling pathway. This is exactly the same as QKI5.

Then explore how QKI5 and lnc6 regulate apoptosis. We found that when suppressing lnc6, the RNA stability of mapk11, mapk12, and mapk14 increased in the p38-MAPK signaling pathway, and when the expression of QKI5 was inhibited, the expression of lnc6 slightly increased compared with the control group; Lnc6 found cleaved caspase3 The expression levels of p-p38, p38 and PARP proteins were lower than those in the control group. While inhibiting both lnc6 and QKI5, the si-lnc6/QKI5 experimental group was also expressed in si-lnc6. Between si-QKI5 groups, the trend was similar to that of NC. The group is roughly the same. These experimental results also confirmed the hypothesis that under normal conditions, QKI5 can maintain the stability of p38-MAPK mRNA and lnc6; when QKI5 is deleted, p38-MAPK mRNA cannot be stabilized by QKI5 and therefore it cannot be correctly translated. Caspase3 expression was inhibited and apoptosis was inhibited. When lnc6 was inhibited, the binding sites of lnc6 and QKI5 were released in large amounts, and the binding sites of QKI5 and p38-MAPK were increased, resulting in the stabilization of p38-MAPK mRNA by QKI5. Cleaved caspase3 expression is activated, which promotes apoptosis. The mRNA of p38-MAPK and lnc6 regulates apoptosis by binding to the binding site of QKI5.

In summary, we investigated the role and mechanism of QKI5 and its related lnc6 in spermatogenesis in vitro: We found that QKI5 can promote apoptosis through activation of p38-MAPK signaling pathway; at the same time, we confirmed that lnc6 can bind to QKI5 and inhibit p38. -MAPK signaling pathway suppresses apoptosis. The mechanism of action of QKI5 and lnc6 may be through competitive regulation of apoptosis regulation.

In the study of apoptosis in QKI5, we also constructed the QKI5 knockout GC1-spg cell line using CRISPR / CAS9 technology. Through screening and identification, 1# targeted single cell line 3 and 2# target sites were finally obtained. For 3 single cell strains, base deletion mutant 1#06 of GTCCGAG was selected as a material for studying cell proliferation and differentiation experiments. The results showed that the cell proliferation of cas-QKI5 experimental group was significantly slower than that of the control group (* P <0.05); the expression levels of c-kit, Mtl5, and Hspa2 markers related to meiosis were detected and found to be significantly lower in the experimental group. Control group. This shows that QKI5 has the function of promoting cell proliferation and cell differentiation.